Increasing capture efficiency of spatial assays

ABSTRACT

This disclosure relates to methods for increasing capture efficiency of a spatial array using rolling circle amplification of a padlock probe that hybridizes to a capture probe. Also provided are methods for using such spatial arrays to detect a biological analyte in a biological sample.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/969,469, filed Feb. 3, 2020. The contents of this application isincorporated herein by reference in its entirety.

BACKGROUND

Cells within a tissue of a subject have differences in cell morphologyand/or function due to varied analyte levels (e.g., gene and/or proteinexpression) within the different cells. The specific position of a cellwithin a tissue (e.g., the cell's position relative to neighboring cellsor the cell's position relative to the tissue microenvironment) canaffect, e.g., the cell's morphology, differentiation, fate, viability,proliferation, behavior, and signaling and cross-talk with other cellsin the tissue.

Spatial heterogeneity has been previously studied using techniques thatonly provide data for a small handful of analytes in the context of anintact tissue or a portion of a tissue, or provide a lot of analyte datafor single cells, but fail to provide information regarding the positionof the single cell in a parent biological sample (e.g., tissue sample).

Genetic material, and related gene and protein expression, influencescellular fate and behavior. The spatial heterogeneity in developingsystems has typically been studied via RNA hybridization,immunohistochemistry, fluorescent reporters, or purification orinduction of pre-defined subpopulations and subsequent genomic profiling(e.g., RNA-seq). Such approaches, however, rely on a small set ofpre-defined markers, therefore introducing selection bias that limitsdiscovery and making it costly and laborious to localize RNAtranscriptome-wide.

SUMMARY

This disclosure relates to methods of increasing capture efficiency of aspatial array. In one aspect, disclosed herein are methods for preparinga spatial array, the method comprising: (a) contacting a substratecomprising a plurality of capture probes with a padlock probe, wherein(i) a capture probe of the plurality of capture probes comprises a firstdocking sequence, a spatial barcode, a capture domain, and a seconddocking sequence; and (ii) the padlock probe comprises: a first dockingpadlock sequence at its 3′ end that is complementary to the firstdocking sequence of the capture probe, and a second docking padlocksequence at its 5′ end that is complementary to the second dockingsequence of the capture probe; (b) hybridizing the first dockingsequence to the first docking padlock sequence and hybridizing thesecond docking sequence to the second docking padlock sequence; (c)ligating the padlock probe, thereby generating a ligated padlock probethat comprises a capture domain; and (d) amplifying the ligated padlockprobe, thereby generating a padlock probe sequence with multiple copiesof the capture domain. In another aspect, provided herein are methods ofincreasing capture efficiency of a spatial array comprising: (a)contacting a substrate comprising a plurality of capture probes with afirst oligonucleotide; wherein (i) a capture probe of the pluralitycomprises a first docking sequence, a second docking sequence, abarcode, and a capture domain; and (ii) the first oligonucleotidecomprises a first complementary sequence complementary to the firstdocking sequence of the capture probe and a second complementarysequence complementary to the second docking sequence of the captureprobe; (b) hybridizing the first docking sequence of the capture probeto the first complementary sequence of the first oligonucleotide andhybridizing the second docking sequence of the capture probe to thesecond complementary sequence of the first oligonucleotide; (c) ligatingthe 3′ end of the first oligonucleotide to the 5′ end of the firstoligonucleotide, thereby forming a ligation product; and (d)amplifyingthe ligation product using rolling circle amplification.

Also provided herein are methods of increasing capture efficiency of aspatial array comprising: (a) contacting a substrate comprising aplurality of capture probes with a first oligonucleotide and a secondoligonucleotide; wherein (i) a capture probe of the plurality comprisesa first docking sequence, a barcode, and a capture domain; (ii) a secondoligonucleotide comprises a second docking sequence; (iii) the firstoligonucleotide comprises a first complementary sequence that iscomplementary to the first docking sequence of the capture probe and asecond complementary sequence that is complementary to the seconddocking sequence of the second oligonucleotide; (b) hybridizing thefirst docking sequence of the capture probe to the first complementarydocking sequence of the first oligonucleotide and hybridizing the seconddocking sequence of the second oligonucleotide to the secondcomplementary docking sequence of the first oligonucleotide; (c)ligating the 3′ end of the first oligonucleotide to the 5′ end of thefirst oligonucleotide, thereby forming a ligation product; and (d)amplifying the ligation product using rolling circle amplification toform an amplified ligation product.

Also provide herein are methods of increasing capture efficiency of aspatial array comprising: (a) contacting a substrate comprising aplurality of capture probes with a first oligonucleotide; wherein (i) acapture probe of the plurality comprises a first docking sequence, asecond docking sequence, a barcode, and a capture domain; (ii) the firstoligonucleotide comprises a first complementary sequence complementaryto the first docking sequence of the capture probe and a secondcomplementary sequence complementary to the first docking sequence ofthe capture probe; (b) hybridizing the first docking sequence and thesecond docking sequence of the capture probe to the first complementarydocking sequence and the second complementary docking sequence of theoligonucleotide; (c) extending the 3′ end of the first oligonucleotideto form an extended 3′ end of the first oligonucleotide; (d) ligatingthe extended 3′ end to the 5′ end of the first oligonucleotide to form aligation product; and (e) amplifying the ligation product using rollingcircle amplification to form an amplified ligation product.

In some instances, the spatial barcode and the capture domain arelocated between the first docking sequence and the second dockingsequence of the capture probe. In some instances, the ligated padlockprobe further comprises a primer sequence, or a complement thereof, anda spatial domain, or a complement thereof. In some instances, theamplifying comprises rolling circle amplification. In some instances,the padlock probe comprises a 3′OH group, wherein the 3′OH group is aprimer for the rolling circle amplification.

In some instances, the step of hybridizing the padlock probe to thecapture probe utilizes a splint oligonucleotide that comprises asequence complementary to the capture probe and a sequence complementaryto the padlock probe.

In some instances, the ligating step further includes extending thepadlock probe via a nucleic acid extension reaction using the captureprobe as a template. In some instances, the extending occurs prior tothe ligating step.

In some instances, the capture domain comprises a poly(T) sequence, arandom sequence, a semi-random sequence or a fixed sequence.

In some instances, the ligation step comprises using enzymatic ligationor chemical ligation. In some instances, the enzymatic ligation utilizesa ligase, wherein the ligase is one or more of a T4 RNA ligase (Rnl2), asplintR ligase, a single stranded DNA ligase, or a T4 DNA ligase.

In some embodiments, a portion of the first oligonucleotide is at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98% or at least 99% complementary to the capture probe. Insome embodiments, a portion of the first oligonucleotide is fullycomplementary to the capture probe. In some embodiments, a portion ofthe second oligonucleotide is at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98% or at least 99%complementary to the capture probe. In some embodiments, a portion ofthe second oligonucleotide is fully complementary to the capture probe.

In some embodiments, a method described herein further comprises copyingthe capture domain sequence using a DNA polymerase.

In some embodiments, a product of the rolling circle amplificationcomprises amplifying multiple copies of a capture sequence and multiplecopies of the spatial barcode.

In some embodiments, the capture sequence is an oligo d(T) sequence. Insome embodiments, the first oligonucleotide comprises a functionalsequence. In some embodiments, the functional sequence is a primersequence. In some embodiments, the first oligonucleotide comprises 3′OHgroup, wherein the 3′OH group is a primer for the rolling circleamplification.

In some embodiments, the second oligonucleotide comprises a functionalsequence. In some embodiments, the functional sequence is a primersequence. In some embodiments, the second oligonucleotide comprises 3′OHgroup, wherein the 3′OH group is a primer for the rolling circleamplification.

In some embodiments, the ligation step comprises using enzymaticligation or chemical ligation. In some embodiments, the enzymaticligation utilizes a ligase. In some embodiments, the ligase is one ormore of a T4 RNA ligase (Rnl2), a splintR ligase, a single stranded DNAligase, or a T4 DNA ligase.

In some embodiments, the amplifying is isothermal. In some embodiments,the amplifying is not isothermal.

In some embodiments, the rolling circle amplification comprises using aPhi29 polymerase.

In some embodiments, a method described herein further comprises a stepof washing away any oligonucleotides that do not hybridize after thehybridizing step.

In some embodiments, a method described herein further comprisesspatially profiling an analyte in a biological sample.

In some embodiments, spatially profiling an analyte in a biologicalsample comprises: (a) contacting the biological sample with thesubstrate comprising the amplified ligation product; (b) releasing theanalyte from the biological sample, wherein the analyte is bound by theamplified ligation product at a distinct spatial position of thesubstrate; (c) detecting the biological analyte bound by the amplifiedligation product; and (d) correlating the biological analyte with thebarcode at the distinct spatial position of the substrate.

In some instances, the methods disclosed herein further includespatially profiling an analyte in a biological sample. In someinstances, spatially profiling the analyte includes the steps of:contacting the spatial array with the biological sample; hybridizing theanalyte or analyte derivative to a capture domain of the multiple copiesof the capture domain; and determining (i) all or a part of the sequenceof the analyte or analyte derivative, or a complement thereof, and (ii)all or a part of the sequence of the spatial barcode, or a complementthereof, and using the determined sequence of (i) and (ii) to determinethe abundance and the location of the analyte in the biological sample.

In some instances, the methods disclosed herein further includecontacting the biological sample with a permeabilization agent, whereinthe permeabilization agent is selected from an organic solvent, adetergent, and an enzyme, or a combination thereof.

In some instances, the determining step comprises sequencing.

In some instances, the methods disclosed herein further include imagingthe biological sample. In some embodiments, the biological sample is atissue section. In some embodiments, the biological sample is aformalin-fixed, paraffin-embedded (FFPE) sample, a frozen sample, or afresh sample. In some embodiments, the biological sample is an FFPEsample.

In some embodiments, the analyte is a DNA molecule or an RNA molecule.In some embodiments, the RNA molecule is mRNA. In some instances, theanalyte is an RNA molecule or a protein, or both.

Also disclosed herein are kits. In some instances, a kit as disclosedherein includes (a) an array comprising a plurality of primers; (b) aplurality of padlock probes; (c) a plurality of enzymes comprising apolymerase and a ligase; and (d) instructions for performing any of themethods disclosed herein.

Also disclosed herein are compositions. In some instances, a compositiondisclosed herein includes (a) a substrate comprising a plurality ofcapture probes, wherein a capture probe of the plurality of captureprobes comprises a first docking sequence, a spatial barcode, a capturedomain, and a second docking sequence; (b) a plurality of amplifiedpadlock probes, wherein an amplified padlock probe of the plurality ofamplified padlock probes comprises: (i) a first docking padlock sequencethat is complementary to the first docking sequence of the captureprobe, (ii) a second docking padlock sequence that is complementary tothe second docking sequence of the capture probe, and (iii) a sequencecomplementary to the capture domain; and wherein the amplified padlockprobe is hybridized to the capture probe. In some instances, an analytehybridized to the amplified padlock probe in the composition. In someinstances, the amplified padlock probe further comprises a primersequence, or a complement thereof, and a spatial domain, or a complementthereof.

All publications, patents, patent applications, and informationavailable on the internet and mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication, patent, patent application, or item of information wasspecifically and individually indicated to be incorporated by reference.To the extent publications, patents, patent applications, and items ofinformation incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

Where values are described in terms of ranges, it should be understoodthat the description includes the disclosure of all possible sub-rangeswithin such ranges, as well as specific numerical values that fallwithin such ranges irrespective of whether a specific numerical value orspecific sub-range is expressly stated.

The term “each,” when used in reference to a collection of items, isintended to identify an individual item in the collection but does notnecessarily refer to every item in the collection, unless expresslystated otherwise, or unless the context of the usage clearly indicatesotherwise.

The singular form “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise. For example, the term “a cell”includes one or more cells, including mixtures thereof “A and/or B” isused herein to include all of the following alternatives: “A”, “B”, “Aor B”, and “A and B”.

Various embodiments of the features of this disclosure are describedherein. However, it should be understood that such embodiments areprovided merely by way of example, and numerous variations, changes, andsubstitutions can occur to those skilled in the art without departingfrom the scope of this disclosure. It should also be understood thatvarious alternatives to the specific embodiments described herein arealso within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

The following drawings illustrate certain embodiments of the featuresand advantages of this disclosure. These embodiments are not intended tolimit the scope of the appended claims in any manner. Like referencesymbols in the drawings indicate like elements.

FIG. 1 is a schematic diagram showing an example of a barcoded captureprobe, as described herein.

FIG. 2 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to target analytes within the sample.

FIG. 3 is a schematic diagram of an exemplary multiplexedspatially-barcoded feature.

FIG. 4 is a schematic diagram of an exemplary analyte capture agent.

FIG. 5 is a schematic diagram depicting an exemplary interaction betweena feature-immobilized capture probe 524 and an analyte capture agent526.

FIGS. 6A-6C are schematics illustrating how streptavidin cell tags canbe utilized in an array-based system to produce a spatially-barcodedcells or cellular contents.

FIG. 7 is a schematic illustrating the generation of a probe using apadlock probe hybridized to a capture probe (top) and a padlock probe, acapture probe, and an oligonucleotide.

FIG. 8 is a schematic showing a padlock probe.

FIG. 9 is a schematic showing the annealing of multiple RNAs to a probe.

DETAILED DESCRIPTION I. Introduction

Spatial analysis methods using capture probes and/or analyte captureagents provide information regarding the abundance and location of ananalyte (e.g., a nucleic acid or protein). The efficiency of spatialanalysis using arrays with capture probes depends, at least in part, onthe density of the probes on the array or the density of the analytescaptured on the array. That is, on how many capture probes can beprinted on the surface of a slide or how many RNA molecules can becaptured. Disclosed herein are methods and compositions for increasingthe efficiency of spatial analysis by increasing the number ofinteractions between the capture probe and the analyte. In this way,analyte detection signal is increased, thus increasing the capturingefficiency, sensitivity, and the resolution of detection on the spatialarray.

Traditionally, these methods identify a singular molecule at a location.Extending these methods to study interactions between two or moreanalytes would provide information on the interactions between two ormore analytes at a location in a biological sample. Analyte captureagents as provided herein comprises an analyte binding moiety affixed toan oligonucleotide. The oligonucleotide comprises a sequence thatuniquely identifies the analyte and moiety. Further, nearbyoligonucleotides affixed to a different moiety in a nearby location canbe ligated to the first oligonucleotide and then can be detected usingthe spatial methods described herein. The methods disclosed herein thusprovide the ability to study the interaction between two or moreanalytes in a biological sample.

Spatial analysis methodologies and compositions described herein canprovide a vast amount of analyte and/or expression data for a variety ofanalytes within a biological sample at high spatial resolution, whileretaining native spatial context. Spatial analysis methods andcompositions can include, e.g., the use of a capture probe including aspatial barcode (e.g., a nucleic acid sequence that provides informationas to the location or position of an analyte within a cell or a tissuesample (e.g., mammalian cell or a mammalian tissue sample) and a capturedomain that is capable of binding to an analyte (e.g., a protein and/ora nucleic acid) produced by and/or present in a cell. Spatial analysismethods and compositions can also include the use of a capture probehaving a capture domain that captures an intermediate agent for indirectdetection of an analyte. For example, the intermediate agent can includea nucleic acid sequence (e.g., a barcode) associated with theintermediate agent. Detection of the intermediate agent is thereforeindicative of the analyte in the cell or tissue sample.

Non-limiting aspects of spatial analysis methodologies and compositionsare described in U.S. Pat. Nos. 10,774,374, 10,724,078, 10,480,022,10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810,9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent ApplicationPublication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641,2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709,2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322,2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875,2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO2020/176788, Rodrigues et al., Science 363(6434):1463-1467, 2019; Lee etal., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gaoet al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol.36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits UserGuide (e.g., Rev C, dated June 2020), and/or the Visium Spatial TissueOptimization Reagent Kits User Guide (e.g., Rev C, dated July 2020),both of which are available at the 10× Genomics Support Documentationwebsite, and can be used herein in any combination. Further non-limitingaspects of spatial analysis methodologies and compositions are describedherein.

Some general terminology that may be used in this disclosure can befound in Section (I)(b) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Typically, a “barcode” is a label, oridentifier, that conveys or is capable of conveying information (e.g.,information about an analyte in a sample, a bead, and/or a captureprobe). A barcode can be part of an analyte, or independent of ananalyte. A barcode can be attached to an analyte. A particular barcodecan be unique relative to other barcodes. For the purpose of thisdisclosure, an “analyte” can include any biological substance,structure, moiety, or component to be analyzed. The term “target” cansimilarly refer to an analyte of interest.

Analytes can be broadly classified into one of two groups: nucleic acidanalytes, and non-nucleic acid analytes. Examples of non-nucleic acidanalytes include, but are not limited to, lipids, carbohydrates,peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins,phosphoproteins, specific phosphorylated or acetylated variants ofproteins, amidation variants of proteins, hydroxylation variants ofproteins, methylation variants of proteins, ubiquitylation variants ofproteins, sulfation variants of proteins, viral proteins (e.g., viralcapsid, viral envelope, viral coat, viral accessory, viralglycoproteins, viral spike, etc.), extracellular and intracellularproteins, antibodies, and antigen binding fragments. In someembodiments, the analyte(s) can be localized to subcellular location(s),including, for example, organelles, e.g., mitochondria, Golgi apparatus,endoplasmic reticulum, chloroplasts, endocytic vesicles, exocyticvesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) canbe peptides or proteins, including without limitation antibodies andenzymes. Additional examples of analytes can be found in Section (I)(c)of WO 2020/176788 and/or U.S. Patent Application Publication No.2020/0277663. In some embodiments, an analyte can be detectedindirectly, such as through detection of an intermediate agent, forexample, a connected probe (e.g., a ligation product) or an analytecapture agent (e.g., an oligonucleotide-conjugated antibody), such asthose described herein.

A “biological sample” is typically obtained from the subject foranalysis using any of a variety of techniques including, but not limitedto, biopsy, surgery, and laser capture microscopy (LCM), and generallyincludes cells and/or other biological material from the subject. Insome embodiments, a biological sample can be a tissue section. In someembodiments, a biological sample can be a fixed and/or stainedbiological sample (e.g., a fixed and/or stained tissue section).Non-limiting examples of stains include histological stains (e.g.,hematoxylin and/or eosin) and immunological stains (e.g., fluorescentstains). In some embodiments, a biological sample (e.g., a fixed and/orstained biological sample) can be imaged. Biological samples are alsodescribed in Section (I)(d) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, a biological sample is permeabilized with one ormore permeabilization reagents. For example, permeabilization of abiological sample can facilitate analyte capture. Exemplarypermeabilization agents and conditions are described in Section(I)(d)(ii)(13) or the Exemplary Embodiments Section of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

Array-based spatial analysis methods involve the transfer of one or moreanalytes from a biological sample to an array of features on asubstrate, where each feature is associated with a unique spatiallocation on the array. Subsequent analysis of the transferred analytesincludes determining the identity of the analytes and the spatiallocation of the analytes within the biological sample. The spatiallocation of an analyte within the biological sample is determined basedon the feature to which the analyte is bound (e.g., directly orindirectly) on the array, and the feature's relative spatial locationwithin the array.

A “capture probe” refers to any molecule capable of capturing (directlyor indirectly) and/or labelling an analyte (e.g., an analyte ofinterest) in a biological sample. In some embodiments, the capture probeis a nucleic acid or a polypeptide. In some embodiments, the captureprobe includes a barcode (e.g., a spatial barcode and/or a uniquemolecular identifier (UMI)) and a capture domain). In some embodiments,a capture probe can include a cleavage domain and/or a functional domain(e.g., a primer-binding site, such as for next-generation sequencing(NGS)).

FIG. 1 is a schematic diagram showing an exemplary capture probe, asdescribed herein. As shown, the capture probe 102 is optionally coupledto a feature 101 by a cleavage domain 103, such as a disulfide linker.The capture probe can include a functional sequence 104 that are usefulfor subsequent processing. The functional sequence 104 can include allor a part of sequencer specific flow cell attachment sequence (e.g., aP5 or P7 sequence), all or a part of a sequencing primer sequence,(e.g., a R1 primer binding site, a R2 primer binding site), orcombinations thereof. The capture probe can also include a spatialbarcode 105. The capture probe can also include a unique molecularidentifier (UMI) sequence 106. While FIG. 1 shows the spatial barcode105 as being located upstream (5′) of UMI sequence 106, it is to beunderstood that capture probes wherein UMI sequence 106 is locatedupstream (5′) of the spatial barcode 105 is also suitable for use in anyof the methods described herein. The capture probe can also include acapture domain 107 to facilitate capture of a target analyte. In someembodiments, the capture probe comprises one or more additionalfunctional sequences that can be located, for example between thespatial barcode 105 and the UMI sequence 106, between the UMI sequence106 and the capture domain 107, or following the capture domain 107. Thecapture domain can have a sequence complementary to a sequence of anucleic acid analyte. The capture domain can have a sequencecomplementary to a connected probe described herein. The capture domaincan have a sequence complementary to a capture handle sequence presentin an analyte capture agent. The capture domain can have a sequencecomplementary to a splint oligonucleotide. Such splint oligonucleotide,in addition to having a sequence complementary to a capture domain of acapture probe, can have a sequence of a nucleic acid analyte, a sequencecomplementary to a portion of a connected probe described herein, and/ora capture handle sequence described herein.

The functional sequences can generally be selected for compatibilitywith any of a variety of different sequencing systems, e.g., Ion TorrentProton or PGM, Illumina sequencing instruments, PacBio, Oxford Nanopore,etc., and the requirements thereof. In some embodiments, functionalsequences can be selected for compatibility with non-commercializedsequencing systems. Examples of such sequencing systems and techniques,for which suitable functional sequences can be used, include (but arenot limited to) Ion Torrent Proton or PGM sequencing, Illuminasequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing.Further, in some embodiments, functional sequences can be selected forcompatibility with other sequencing systems, includingnon-commercialized sequencing systems.

In some embodiments, the spatial barcode 105 and functional sequences104 is common to all of the probes attached to a given feature. In someembodiments, the UMI sequence 106 of a capture probe attached to a givenfeature is different from the UMI sequence of a different capture probeattached to the given feature.

FIG. 2 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to analytes within the sample. The capture probe 201 contains acleavage domain 202, a cell penetrating peptide 203, a reporter molecule204, and a disulfide bond (—S—S—). 205 represents all other parts of acapture probe, for example a spatial barcode and a capture domain.

FIG. 3 is a schematic diagram of an exemplary multiplexedspatially-barcoded feature. In FIG. 3, the feature 301 can be coupled tospatially-barcoded capture probes, wherein the spatially-barcoded probesof a particular feature can possess the same spatial barcode, but havedifferent capture domains designed to associate the spatial barcode ofthe feature with more than one target analyte. For example, a featuremay be coupled to four different types of spatially-barcoded captureprobes, each type of spatially-barcoded capture probe possessing thespatial barcode 302. One type of capture probe associated with thefeature includes the spatial barcode 302 in combination with a poly(T)capture domain 303, designed to capture mRNA target analytes. A secondtype of capture probe associated with the feature includes the spatialbarcode 302 in combination with a random N-mer capture domain 304 forgDNA analysis. A third type of capture probe associated with the featureincludes the spatial barcode 302 in combination with a capture domaincomplementary to a capture handle sequence of an analyte capture agentof interest 305. A fourth type of capture probe associated with thefeature includes the spatial barcode 302 in combination with a capturedomain that can specifically bind a nucleic acid molecule 306 that canfunction in a CRISPR assay (e.g., CRISPR/Cas9). While only fourdifferent capture probe-barcoded constructs are shown in FIG. 3,capture-probe barcoded constructs can be tailored for analyses of anygiven analyte associated with a nucleic acid and capable of binding withsuch a construct. For example, the schemes shown in FIG. 3 can also beused for concurrent analysis of other analytes disclosed herein,including, but not limited to: (a) mRNA, a lineage tracing construct,cell surface or intracellular proteins and metabolites, and gDNA; (b)mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq)cell surface or intracellular proteins and metabolites, and aperturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein); (c)mRNA, cell surface or intracellular proteins and/or metabolites, abarcoded labelling agent (e.g., the MHC multimers described herein), anda V(D)J sequence of an immune cell receptor (e.g., T-cell receptor). Insome embodiments, a perturbation agent can be a small molecule, anantibody, a drug, an aptamer, a miRNA, a physical environmental (e.g.,temperature change), or any other known perturbation agents. See, e.g.,Section (II)(b) (e.g., subsections (i)-(vi)) of WO 2020/176788 and/orU.S. Patent Application Publication No. 2020/0277663. Generation ofcapture probes can be achieved by any appropriate method, includingthose described in Section (II)(d)(ii) of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663.

In some embodiments, more than one analyte type (e.g., nucleic acids andproteins) from a biological sample can be detected (e.g., simultaneouslyor sequentially) using any appropriate multiplexing technique, such asthose described in Section (IV) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, detection of one or more analytes (e.g., proteinanalytes) can be performed using one or more analyte capture agents. Asused herein, an “analyte capture agent” refers to an agent thatinteracts with an analyte (e.g., an analyte in a biological sample) andwith a capture probe (e.g., a capture probe attached to a substrate or afeature) to identify the analyte. In some embodiments, the analytecapture agent includes: (i) an analyte binding moiety (e.g., that bindsto an analyte), for example, an antibody or antigen-binding fragmentthereof; (ii) analyte binding moiety barcode; and (iii) a capture handlesequence. As used herein, the term “analyte binding moiety barcode”refers to a barcode that is associated with or otherwise identifies theanalyte binding moiety. As used herein, the term “analyte capturesequence” or “capture handle sequence” refers to a region or moietyconfigured to hybridize to, bind to, couple to, or otherwise interactwith a capture domain of a capture probe. In some embodiments, a capturehandle sequence is complementary to a capture domain of a capture probe.In some cases, an analyte binding moiety barcode (or portion thereof)may be able to be removed (e.g., cleaved) from the analyte captureagent.

FIG. 4 is a schematic diagram of an exemplary analyte capture agent 402comprised of an analyte-binding moiety 404 and an analyte-binding moietybarcode domain 408. The exemplary analyte-binding moiety 404 is amolecule capable of binding to an analyte 406 and the analyte captureagent is capable of interacting with a spatially-barcoded capture probe.The analyte-binding moiety can bind to the analyte 406 with highaffinity and/or with high specificity. The analyte capture agent caninclude an analyte-binding moiety barcode domain 408, a nucleotidesequence (e.g., an oligonucleotide), which can hybridize to at least aportion or an entirety of a capture domain of a capture probe. Theanalyte-binding moiety barcode domain 408 can comprise an analytebinding moiety barcode and a capture handle sequence described herein.The analyte-binding moiety 404 can include a polypeptide and/or anaptamer. The analyte-binding moiety 404 can include an antibody orantibody fragment (e.g., an antigen-binding fragment).

FIG. 5 is a schematic diagram depicting an exemplary interaction betweena feature-immobilized capture probe 524 and an analyte capture agent526. The feature-immobilized capture probe 524 can include a spatialbarcode 508 as well as functional sequences 506 and UMI 510, asdescribed elsewhere herein. The capture probe can also include a capturedomain 512 that is capable of binding to an analyte capture agent 526.The analyte capture agent 526 can include a functional sequence 518,analyte binding moiety barcode 516, and a capture handle sequence 514that is capable of binding to the capture domain 512 of the captureprobe 524. The analyte capture agent can also include a linker 520 thatallows the capture agent barcode domain 516 to couple to the analytebinding moiety 522.

FIGS. 6A, 6B, and 6C are schematics illustrating how streptavidin celltags can be utilized in an array-based system to produce aspatially-barcoded cell or cellular contents. For example, as shown inFIG. 6A, peptide-bound major histocompatibility complex (MHC) can beindividually associated with biotin ((β2m) and bound to a streptavidinmoiety such that the streptavidin moiety comprises multiple pMHCmoieties. Each of these moieties can bind to a TCR such that thestreptavidin binds to a target T-cell via multiple MCH/TCR bindinginteractions. Multiple interactions synergize and can substantiallyimprove binding affinity. Such improved affinity can improve labellingof T-cells and also reduce the likelihood that labels will dissociatefrom T-cell surfaces. As shown in FIG. 6B, a capture agent barcodedomain 601 can be modified with streptavidin 602 and contacted withmultiple molecules of biotinylated MHC 603 such that the biotinylatedMHC 603 molecules are coupled with the streptavidin conjugated captureagent barcode domain 601. The result is a barcoded MHC multimer complex605. As shown in FIG. 6B, the capture agent barcode domain sequence 601can identify the MHC as its associated label and also includes optionalfunctional sequences such as sequences for hybridization with otheroligonucleotides. As shown in FIG. 6C, one example oligonucleotide iscapture probe 606 that comprises a complementary sequence (e.g., rGrGrGcorresponding to C C C), a barcode sequence and other functionalsequences, such as, for example, a UMI, an adapter sequence (e.g.,comprising a sequencing primer sequence (e.g., R1 or a partial R1(“pR1”), R2), a flow cell attachment sequence (e.g., P5 or P7 or partialsequences thereof)), etc. In some cases, capture probe 606 may at firstbe associated with a feature (e.g., a gel bead) and released from thefeature. In other embodiments, capture probe 606 can hybridize with acapture agent barcode domain 601 of the MHC-oligonucleotide complex 605.The hybridized oligonucleotides (Spacer C C C and Spacer rGrGrG) canthen be extended in primer extension reactions such that constructscomprising sequences that correspond to each of the two spatial barcodesequences (the spatial barcode associated with the capture probe, andthe barcode associated with the MHC-oligonucleotide complex) aregenerated. In some cases, one or both of these corresponding sequencesmay be a complement of the original sequence in capture probe 606 orcapture agent barcode domain 601. In other embodiments, the captureprobe and the capture agent barcode domain are ligated together. Theresulting constructs can be optionally further processed (e.g., to addany additional sequences and/or for clean-up) and subjected tosequencing. As described elsewhere herein, a sequence derived from thecapture probe 606 spatial barcode sequence may be used to identify afeature and the sequence derived from spatial barcode sequence on thecapture agent barcode domain 601 may be used to identify the particularpeptide MHC complex 604 bound on the surface of the cell (e.g., whenusing MHC-peptide libraries for screening immune cells or immune cellpopulations).

Additional description of analyte capture agents can be found in Section(II)(b)(ix) of WO 2020/176788 and/or Section (II)(b)(viii) U.S. PatentApplication Publication No. 2020/0277663.

There are at least two methods to associate a spatial barcode with oneor more neighboring cells, such that the spatial barcode identifies theone or more cells, and/or contents of the one or more cells, asassociated with a particular spatial location. One method is to promoteanalytes or analyte proxies (e.g., intermediate agents) out of a celland towards a spatially-barcoded array (e.g., includingspatially-barcoded capture probes). Another method is to cleavespatially-barcoded capture probes from an array and promote thespatially-barcoded capture probes towards and/or into or onto thebiological sample.

In some cases, capture probes may be configured to prime, replicate, andconsequently yield optionally barcoded extension products from atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent (e.g., a connected probe (e.g., a ligation product)or an analyte capture agent), or a portion thereof), or derivativesthereof (see, e.g., Section (II)(b)(vii) of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663 regarding extendedcapture probes). In some cases, capture probes may be configured to forma connected probe (e.g., a ligation product) with a template (e.g., aDNA or RNA template, such as an analyte or an intermediate agent, orportion thereof), thereby creating ligation products that serve asproxies for a template.

As used herein, an “extended capture probe” refers to a capture probehaving additional nucleotides added to the terminus (e.g., 3′ or 5′ end)of the capture probe thereby extending the overall length of the captureprobe. For example, an “extended 3′ end” indicates additionalnucleotides were added to the most 3′ nucleotide of the capture probe toextend the length of the capture probe, for example, by polymerizationreactions used to extend nucleic acid molecules including templatedpolymerization catalyzed by a polymerase (e.g., a DNA polymerase or areverse transcriptase). In some embodiments, extending the capture probeincludes adding to a 3′ end of a capture probe a nucleic acid sequencethat is complementary to a nucleic acid sequence of an analyte orintermediate agent bound to the capture domain of the capture probe. Insome embodiments, the capture probe is extended using reversetranscription. In some embodiments, the capture probe is extended usingone or more DNA polymerases. The extended capture probes include thesequence of the capture probe and the sequence of the spatial barcode ofthe capture probe.

In some embodiments, extended capture probes are amplified (e.g., inbulk solution or on the array) to yield quantities that are sufficientfor downstream analysis, e.g., via DNA sequencing. In some embodiments,extended capture probes (e.g., DNA molecules) act as templates for anamplification reaction (e.g., a polymerase chain reaction).

Additional variants of spatial analysis methods, including in someembodiments, an imaging step, are described in Section (II)(a) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.Analysis of captured analytes (and/or intermediate agents or portionsthereof), for example, including sample removal, extension of captureprobes, sequencing (e.g., of a cleaved extended capture probe and/or acDNA molecule complementary to an extended capture probe), sequencing onthe array (e.g., using, for example, in situ hybridization or in situligation approaches), temporal analysis, and/or proximity capture, isdescribed in Section (II)(g) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663. Some quality control measuresare described in Section (II)(h) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Spatial information can provide information of biological and/or medicalimportance. For example, the methods and compositions described hereincan allow for: identification of one or more biomarkers (e.g.,diagnostic, prognostic, and/or for determination of efficacy of atreatment) of a disease or disorder; identification of a candidate drugtarget for treatment of a disease or disorder; identification (e.g.,diagnosis) of a subject as having a disease or disorder; identificationof stage and/or prognosis of a disease or disorder in a subject;identification of a subject as having an increased likelihood ofdeveloping a disease or disorder; monitoring of progression of a diseaseor disorder in a subject; determination of efficacy of a treatment of adisease or disorder in a subject; identification of a patientsubpopulation for which a treatment is effective for a disease ordisorder; modification of a treatment of a subject with a disease ordisorder; selection of a subject for participation in a clinical trial;and/or selection of a treatment for a subject with a disease ordisorder.

Spatial information can provide information of biological importance.For example, the methods and compositions described herein can allowfor: identification of transcriptome and/or proteome expression profiles(e.g., in healthy and/or diseased tissue); identification of multipleanalyte types in close proximity (e.g., nearest neighbor analysis);determination of up- and/or down-regulated genes and/or proteins indiseased tissue; characterization of tumor microenvironments;characterization of tumor immune responses; characterization of cellstypes and their co-localization in tissue; and identification of geneticvariants within tissues (e.g., based on gene and/or protein expressionprofiles associated with specific disease or disorder biomarkers).

Typically, for spatial array-based methods, a substrate functions as asupport for direct or indirect attachment of capture probes to featuresof the array. A “feature” is an entity that acts as a support orrepository for various molecular entities used in spatial analysis. Insome embodiments, some or all of the features in an array arefunctionalized for analyte capture. Exemplary substrates are describedin Section (II)(c) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Exemplary features and geometricattributes of an array can be found in Sections (II)(d)(i),(II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Generally, analytes and/or intermediate agents (or portions thereof) canbe captured when contacting a biological sample with a substrateincluding capture probes (e.g., a substrate with capture probesembedded, spotted, printed, fabricated on the substrate, or a substratewith features (e.g., beads, wells) comprising capture probes). As usedherein, “contact,” “contacted,” and/or “contacting,” a biological samplewith a substrate refers to any contact (e.g., direct or indirect) suchthat capture probes can interact (e.g., bind covalently ornon-covalently (e.g., hybridize)) with analytes from the biologicalsample. Capture can be achieved actively (e.g., using electrophoresis)or passively (e.g., using diffusion). Analyte capture is furtherdescribed in Section (II)(e) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by attaching and/orintroducing a molecule (e.g., a peptide, a lipid, or a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) to a biologicalsample (e.g., to a cell in a biological sample). In some embodiments, aplurality of molecules (e.g., a plurality of nucleic acid molecules)having a plurality of barcodes (e.g., a plurality of spatial barcodes)are introduced to a biological sample (e.g., to a plurality of cells ina biological sample) for use in spatial analysis. In some embodiments,after attaching and/or introducing a molecule having a barcode to abiological sample, the biological sample can be physically separated(e.g., dissociated) into single cells or cell groups for analysis. Somesuch methods of spatial analysis are described in Section (III) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by detecting multipleoligonucleotides that hybridize to an analyte. In some instances, forexample, spatial analysis can be performed using RNA-templated ligation(RTL). Methods of RTL have been described previously. See, e.g., Credleet al., Nucleic Acids Res. 2017 Aug. 21; 45(14):e128. Typically, RTLincludes hybridization of two oligonucleotides to adjacent sequences onan analyte (e.g., an RNA molecule, such as an mRNA molecule). In someinstances, the oligonucleotides are DNA molecules. In some instances,one of the oligonucleotides includes at least two ribonucleic acid basesat the 3′ end and/or the other oligonucleotide includes a phosphorylatednucleotide at the 5′ end. In some instances, one of the twooligonucleotides includes a capture domain (e.g., a poly(A) sequence, anon-homopolymeric sequence). After hybridization to the analyte, aligase (e.g., SplintR ligase) ligates the two oligonucleotides together,creating a connected probe (e.g., a ligation product). In someinstances, the two oligonucleotides hybridize to sequences that are notadjacent to one another. For example, hybridization of the twooligonucleotides creates a gap between the hybridized oligonucleotides.In some instances, a polymerase (e.g., a DNA polymerase) can extend oneof the oligonucleotides prior to ligation. After ligation, the connectedprobe (e.g., a ligation product) is released from the analyte. In someinstances, the connected probe (e.g., a ligation product) is releasedusing an endonuclease (e.g., RNAse H). The released connected probe(e.g., a ligation product) can then be captured by capture probes (e.g.,instead of direct capture of an analyte) on an array, optionallyamplified, and sequenced, thus determining the location and optionallythe abundance of the analyte in the biological sample.

During analysis of spatial information, sequence information for aspatial barcode associated with an analyte is obtained, and the sequenceinformation can be used to provide information about the spatialdistribution of the analyte in the biological sample. Various methodscan be used to obtain the spatial information. In some embodiments,specific capture probes and the analytes they capture are associatedwith specific locations in an array of features on a substrate. Forexample, specific spatial barcodes can be associated with specific arraylocations prior to array fabrication, and the sequences of the spatialbarcodes can be stored (e.g., in a database) along with specific arraylocation information, so that each spatial barcode uniquely maps to aparticular array location.

Alternatively, specific spatial barcodes can be deposited atpredetermined locations in an array of features during fabrication suchthat at each location, only one type of spatial barcode is present sothat spatial barcodes are uniquely associated with a single feature ofthe array. Where necessary, the arrays can be decoded using any of themethods described herein so that spatial barcodes are uniquelyassociated with array feature locations, and this mapping can be storedas described above.

When sequence information is obtained for capture probes and/or analytesduring analysis of spatial information, the locations of the captureprobes and/or analytes can be determined by referring to the storedinformation that uniquely associates each spatial barcode with an arrayfeature location. In this manner, specific capture probes and capturedanalytes are associated with specific locations in the array offeatures. Each array feature location represents a position relative toa coordinate reference point (e.g., an array location, a fiducialmarker) for the array. Accordingly, each feature location has an“address” or location in the coordinate space of the array.

Some exemplary spatial analysis workflows are described in the ExemplaryEmbodiments section of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. See, for example, the Exemplary embodimentstarting with “In some non-limiting examples of the workflows describedherein, the sample can be immersed . . . ” of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663. See also, e.g., theVisium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C,dated June 2020), and/or the Visium Spatial Tissue Optimization ReagentKits User Guide (e.g., Rev C, dated July 2020).

In some embodiments, spatial analysis can be performed using dedicatedhardware and/or software, such as any of the systems described inSections (II)(e)(ii) and/or (V) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663, or any of one or more of thedevices or methods described in Sections Control Slide for Imaging,Methods of Using Control Slides and Substrates for, Systems of UsingControl Slides and Substrates for Imaging, and/or Sample and ArrayAlignment Devices and Methods, Informational labels of WO 2020/123320.

Suitable systems for performing spatial analysis can include componentssuch as a chamber (e.g., a flow cell or sealable, fluid-tight chamber)for containing a biological sample. The biological sample can be mountedfor example, in a biological sample holder. One or more fluid chamberscan be connected to the chamber and/or the sample holder via fluidconduits, and fluids can be delivered into the chamber and/or sampleholder via fluidic pumps, vacuum sources, or other devices coupled tothe fluid conduits that create a pressure gradient to drive fluid flow.One or more valves can also be connected to fluid conduits to regulatethe flow of reagents from reservoirs to the chamber and/or sampleholder.

The systems can optionally include a control unit that includes one ormore electronic processors, an input interface, an output interface(such as a display), and a storage unit (e.g., a solid state storagemedium such as, but not limited to, a magnetic, optical, or other solidstate, persistent, writeable and/or re-writeable storage medium). Thecontrol unit can optionally be connected to one or more remote devicesvia a network. The control unit (and components thereof) can generallyperform any of the steps and functions described herein. Where thesystem is connected to a remote device, the remote device (or devices)can perform any of the steps or features described herein. The systemscan optionally include one or more detectors (e.g., CCD, CMOS) used tocapture images. The systems can also optionally include one or morelight sources (e.g., LED-based, diode-based, lasers) for illuminating asample, a substrate with features, analytes from a biological samplecaptured on a substrate, and various control and calibration media.

The systems can optionally include software instructions encoded and/orimplemented in one or more of tangible storage media and hardwarecomponents such as application specific integrated circuits. Thesoftware instructions, when executed by a control unit (and inparticular, an electronic processor) or an integrated circuit, can causethe control unit, integrated circuit, or other component executing thesoftware instructions to perform any of the method steps or functionsdescribed herein.

In some cases, the systems described herein can detect (e.g., registeran image) the biological sample on the array. Exemplary methods todetect the biological sample on an array are described in PCTApplication No. 2020/061064 and/or U.S. patent application Ser. No.16/951,854.

Prior to transferring analytes from the biological sample to the arrayof features on the substrate, the biological sample can be aligned withthe array. Alignment of a biological sample and an array of featuresincluding capture probes can facilitate spatial analysis, which can beused to detect differences in analyte presence and/or level withindifferent positions in the biological sample, for example, to generate athree-dimensional map of the analyte presence and/or level. Exemplarymethods to generate a two- and/or three-dimensional map of the analytepresence and/or level are described in PCT Application No. 2020/053655and spatial analysis methods are generally described in WO 2020/061108and/or U.S. patent application Ser. No. 16/951,864.

In some cases, a map of analyte presence and/or level can be aligned toan image of a biological sample using one or more fiducial markers,e.g., objects placed in the field of view of an imaging system whichappear in the image produced, as described in the Substrate AttributesSection, Control Slide for Imaging Section of WO 2020/123320, PCTApplication No. 2020/061066, and/or U.S. patent application Ser. No.16/951,843. Fiducial markers can be used as a point of reference ormeasurement scale for alignment (e.g., to align a sample and an array,to align two substrates, to determine a location of a sample or array ona substrate relative to a fiducial marker) and/or for quantitativemeasurements of sizes and/or distances.

The sandwich process is described in PCT Patent Application PublicationNo. WO 2020/123320, which is incorporated by reference in its entirety.

II. Spatial Analytical Methodology and Increasing Capture Efficiency (a)Introduction

Provided herein are methods of increasing capture efficiency of aspatial array, e.g., a spatial array as described herein. In someembodiments, a method of increasing capture efficiency of a spatialarray includes hybridizing a capture probe, e.g., any of the captureprobes described herein, to an oligonucleotide. As disclosed herein, thecapture probe comprises two sequences to which an oligonucleotide (i.e.,a padlock probe) hybridizes. In some instances, in between the twosequences to which an oligonucleotide (i.e., a padlock probe) hybridizesis a capture domain sequence. In some instances, the oligonucleotide canbe a padlock probe. A “padlock probe,” (also referred to as a padlockoligonucleotide, second probe, or second oligonucleotide) or as referredto herein, is an oligonucleotide that has, at its 5′ and 3′ ends,sequences that are complementary or nearly complementary to adjacent ornearby target sequences on a capture probe. Upon hybridization to thetarget sequences, the two ends of the padlock probe are either broughtinto contact or an end is extended until the two ends are brought intocontact, allowing circularization of the padlock probe by ligation. Insome embodiments, circularization of the padlock probe is performedusing a ligation step. In some embodiments, a ligase is used. In someembodiments, the hybridized padlock probe is circularized using apolymerase (e.g., a DNA polymerase). See, for example, U.S. Pat. No.7,358,047; Larsson et al. Nat Methods. 2004 December; 1(3):227-32;Larsson et al. Nat Methods. 2010 May; 7(5):395-7; and Ke et al. NatMethods. 2013 September; 10(9):857-60; each of which is incorporatedherein by reference in its entirety. After circularization of thepadlock oligonucleotide, rolling circle amplification, for example, canbe used to amplify the ligation product (see, for example, FIG. 7). Suchmethods can be useful for generating probes able to bind to more thanone analyte (e.g., the probe can have more than one capture domain).Upon amplification, the sequence comprises multiple capture probesequences to which analytes can bind. Thus, the methods disclosed hereinprovide the ability to capture a greater number of analytes in a givenarea of a biological sample compared to a capture probe comprising onecapture domain.

Accordingly, a method of increasing capture efficiency of a spatialarray can include contacting a substrate including a plurality ofcapture probes with a first oligonucleotide; wherein a capture probe ofthe plurality includes a first docking sequence, a second dockingsequence, a barcode, and a capture domain; and the first oligonucleotideincludes a first sequence complementary to the first docking sequence ofthe capture probe and a second sequence complementary to the seconddocking sequence of the capture probe; hybridizing the first dockingsequence of the capture probe to the first complementary sequence of thefirst oligonucleotide and hybridizing the second docking sequence of thecapture probe to the second complementary sequence of the firstoligonucleotide; ligating the 3′ end of the first oligonucleotide to the5′ end of the first oligonucleotide, thereby forming a circularizedligation product; and amplifying the ligation product usingamplification (e.g., rolling circle amplification). In some embodiments,the plurality of capture probes are immobilized on the substrate.

In some embodiments, the methods disclosed herein include use of asplint oligonucleotide. In some embodiments, the splint oligonucleotidehybridizes to both the padlock probe and the capture probe on the array.See, for example, FIG. 7. In some embodiments, the secondoligonucleotide includes sequences that are complementary (partially orfully) to a capture probe, a padlock probe, or both. In someembodiments, the second oligonucleotide facilitates hybridization of thefirst oligonucleotide to the capture probe. After hybridization, of thepadlock probe to the capture probe and the splint oligonucleotide,ligation and amplification proceeds, thereby providing capture of agreater number of analytes in a given area of a biological samplecompared to a capture probe comprising one capture domain.

Additional embodiments are provided herein.

(b) Compositions for Increasing Capture Efficiency of a Spatial Array

1. Capture Probes and Arrays

Disclosed herein are capture probes that detect (e.g., hybridize to)analytes in a biological sample. In some instances, the capture probescomprise a plurality of nucleotides. In some instances, the captureprobes comprise DNA.

In some instances, capture probes are placed on the substrate. In someinstances, the capture probes are affixed to the surface of a substrate.In some instances, the capture probes are affixed to the surface of asubstrate at the 5′ end of each capture probe. In some instances, the 3′end of the capture probe is distal to the substrate.

The length of a capture probe as disclosed herein can vary. In someinstances, the length of the capture probe is from 50 nucleotides to 500nucleotides (e.g., about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Insome instances, the length of the capture probe is from 500 to 1000nucleotides.

In some instances, the capture probe comprises a first docking sequence,a capture domain, and a second docking sequence. In some embodiments,the first docking sequence of the capture probe is within about 5 toabout 200 nucleotides of the second docking sequence of the captureprobe. For example, the first docking sequence of the capture probe iswithin about 5 to about 10, about 5 to about 20, about 5 to about 30,about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5to about 70, about 5 to about 80, about 5 to about 90, about 90 to about100, about 80 to about 100, about 70 to about 100, about 60 to about100, about 50 to about 200, about 40 to about 200, about 30 to about200, about 20 to about 200, or about 10 to about 200 nucleotides to thesecond docking sequence of the capture probe.

In some embodiments, after hybridization of the first oligonucleotide tothe capture probe, there can be a gap between the first complementarysequence and the second complementary sequence (see, for example, FIG.8). For example, the capture probe can include one or more other domainsbetween the first docking sequence and the second docking sequence. Ininstances in which the first docking sequence and the second dockingsequence on the capture probe are not adjacent to one another, thecapture probe include a capture domain between the two dockingsequences. In some instances, the capture domain indiscriminatelycaptures analytes of interest. For example, in some instances, thecapture domain comprises a sequence that is complementary to apoly-adenylated sequence of an mRNA molecule. In some instances, thecapture domain comprises a poly-thymine (i.e., poly(T)) sequence. Insome instances, the poly(T) sequence is about 10 to about 100nucleotides in length. It is appreciated that the sequence of thecapture domain should be long enough in order to capture an analyte.

In some instance, the capture domain is designed to detect one or morespecific analytes of interest. For example, a capture domain can bedesigned so that it comprises a sequence that is complementary orsubstantially complementary to one analyte of interest. Thus, thepresence of a single analyte can be detected. Alternatively, the capturedomain can be designed so that it comprises a sequence that iscomplementary or substantially complementary to a conserved region ofmultiple related analytes. In some instances, the multiple relatedanalytes are analytes that function in the same or similar cellularpathways or that have conserved homology and/or function. The design ofthe capture probe can be determined based on the intent of the user andcan be any sequence that can be used to detect an analyte of interest.In some embodiments, the capture domain sequence can therefore berandom, semi-random, defined or combinations thereof, depending on thetarget analyte(s) of interest.

In some embodiments, the first docking sequence of the capture probe isadjacent to the second docking sequence of the capture probe. Forexample, in some embodiments, the first complementary sequence of thefirst oligonucleotide and the second complementary sequence of the firstoligonucleotide hybridize to a portion of the capture probe such thatthe first complementary sequence of the first oligonucleotide and thesecond complementary sequence of the first oligonucleotide are withinabout 1, about 2, about 3, about 4, or about 5, about 6, about 7, about8, about 9, about 10, about 15, about 20, about 30, about 40, about 50,about 60, about 70, about 80, about 90, about 100, about 125, about 150,about 175, about 200, about 250, about 300, about 350, about 400, about450, about 500, or more nucleotides of each other on the capture probe.In instances in which the first docking sequence and the second dockingsequence are adjacent to one another, the capture domain can be locatedon the padlock probe, discussed below.

In some instances, the capture probes includes one or more additionalsequences. In some instances, the capture probe comprises a spatialbarcode. In some instances, the spatial barcode 5′ to the first dockingsite. In some instances, the spatial barcode is 3′ to the second dockingsite. In some instances, the spatial barcode is between the firstdocking site and the second docking site on the capture probe. Thespatial barcode is a unique sequence that provides the location of thecapture probe (and ultimately the analyte in the biological sample).

In some instances, the capture probe comprises one or more functionaldomains. For instance, in some embodiments, the capture probe includes aunique molecular identifier. A unique molecular identifier is acontiguous nucleic acid segment or two or more non-contiguous nucleicacid segments that function as a label or identifier for a particularanalyte, or for a capture probe that binds a particular analyte (e.g.,via the capture domain). The UMI can include from about 6 to about 20 ormore nucleotides within the sequence of the capture probes. In someembodiments, the length of a UMI sequence can be about 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In someembodiments, the length of a UMI sequence can be at least about 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. Insome embodiments, the length of a UMI sequence is at most about 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter.

In some instances, the capture probe comprises a cleavage domain. Thecleavage domain represents the portion of the probe that is used toreversibly attach the probe to an array feature, as will be describedfurther herein. Further, one or more segments or regions of the captureprobe can optionally be released from the array feature by cleavage ofthe cleavage domain. As an example, spatial barcodes and/or universalmolecular identifiers (UMIs) can be released by cleavage of the cleavagedomain.

In some embodiments, a cleavage domain is absent from the capture probe.Examples of substrates with attached capture probes lacking a cleavagedomain are described for example in Macosko et al., (2015) Cell 161,1202-1214, the entire contents of which are incorporated herein byreference.

In some embodiments, the region of the capture probe corresponding tothe cleavage domain can be used for some other function. For example, anadditional region for nucleic acid extension or amplification can beincluded where the cleavage domain would normally be positioned. In suchembodiments, the region can supplement the functional domain or evenexist as an additional functional domain. In some embodiments, thecleavage domain is present but its use is optional.

In some instances, the capture probe comprises any combination of any ofthe above-described sequences.

2. Padlock Probes

Also disclosed herein is a padlock probe (or padlock oligonucleotide,second probe, or second nucleotide). The padlock probe includes aplurality of nucleotides. In some instances, the padlock probe comprisesDNA.

The length of a padlock probe as disclosed herein can vary. In someinstances, the length of the padlock probe is from 50 nucleotides to 500nucleotides (e.g., about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Insome instances, the length of the padlock probe is from 500 to 1000nucleotides.

Referring to FIG. 8, the padlock probe comprises from 3′ to 5′, asequence 801 complementary (or substantially complementary) to the firstdocking sequence, a padlock sequence 802, and a sequence 803complementary (or substantially complementary) to the second dockingsequence. In some instances, the sequence 801 is called the firstdocking padlock sequence. In some instances, the sequence 803 is calledthe second docking padlock sequence.

In some instances, the first docking padlock sequence comprises asequence that is complementary to a portion of the first dockingsequence of the capture probe. In some instances, the first dockingpadlock sequences is from 10 to 100 nucleotides in length. In someinstances, the first docking padlock sequence is about 75%, 80%, 85%,90%, 95%, or 100% complementary to the first docking sequence of thecapture probe. The first docking padlock sequence can be designed usingany sequence of interest so long as hybridization is achieved. In someinstances, the first docking padlock sequence (and its complementarysequence) is not found in the genome of the organism of interest (e.g.,not found in human, mouse, rat genome, etc.). One skilled in the art candesign a sequence so that off-targets are not bound, or notsubstantially bound, to the first docking padlock sequence.

In some instances, the second docking padlock sequence comprises asequence that is complementary to a portion of the second dockingsequence of the capture probe. In some instances, the second dockingpadlock sequences is from 10 to 100 nucleotides in length. In someinstances, the second docking padlock sequence is about 75%, 80%, 85%,90%, 95%, or 100% complementary to the second docking sequence of thecapture probe. The second docking padlock sequence can be designed usingany sequence of interest so long as hybridization is achieved. In someinstances, the second docking padlock sequence (and its complementarysequence) is not found in the genome of the organism of interest (e.g.,not found in human, mouse, rat genome, etc.). One skilled in the art candesign a sequence so that off-targets are not bound, or notsubstantially bound, to the second docking padlock sequence.

In some instances, the padlock sequence 802 is a DNA sequence. In someinstances, the padlock sequence 802 is about 50 to 500 nucleotides inlength (i.e., about 50, about 100, about 150, about 200, about 250,about 300, about 350, about 400, about 450, about 500). In someinstances, the padlock sequence 802 can be designed to include one ormore functional sequences. For instance, in some embodiments, thepadlock sequence 802 comprises a capture domain sequence (e.g., apoly(T) sequence or a random, semi-random, or defined sequence asdescribed above). In some instances, the capture domain sequence of thepadlock is utilized when the first docking sequence and the seconddocking sequence are adjacent to one another on the capture probe. Insome instances, the padlock sequence 802 comprises a primer sequencethat can be used for amplification of the padlock probe. In someinstances, the padlock sequence 802 comprises a cleaving domain aspreviously described.

In some embodiments, the padlock probe includes a functional sequence,e.g., any of the functional sequences described herein. In someembodiments, the padlock probe includes a 3′OH group. In someembodiments, the 3′OH group is a primer for the rolling circleamplification.

3. Splint Oligonucleotides

In some instances, a splint oligonucleotide is used to facilitatehybridization of the padlock probe to the capture probe. Referring toFIG. 7 (bottom panel), in some instances, the splint oligonucleotidecomprises sequences that are complementary to the capture probe and tothe padlock probe. In some instances, a portion of the splintoligonucleotide is complementary to a portion of the capture probe. Insome instances, a portion of the splint oligonucleotide is complementaryto a portion of the padlock probe. In some instances, the splintoligonucleotide comprises a sequence that is complementary to the firstdocking padlock sequence. In some instance, the splint oligonucleotidecomprises a sequence that is complementary to the second docking padlocksequence.

In some embodiments, a portion of the splint oligonucleotide is at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98% or at least 99% complementary to the capture probe. Insome embodiments, a portion of the splint oligonucleotide is fullycomplementary to the capture probe.

In some embodiments, a portion of the splint oligonucleotide is at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98% or at least 99% complementary to the first dockingpadlock sequence. In some embodiments, a portion of the splintoligonucleotide is fully complementary to the first docking padlocksequence.

In some embodiments, a portion of the splint oligonucleotide is at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98% or at least 99% complementary to the second dockingpadlock sequence. In some embodiments, a portion of the splintoligonucleotide is fully complementary to the second docking padlocksequence.

The splint oligonucleotide includes a plurality of nucleotides. In someinstances, the splint oligonucleotide comprises DNA.

The length of a splint oligonucleotide as disclosed herein can vary. Insome instances, the length of the splint oligonucleotide is from 50nucleotides to 500 nucleotides (e.g., about 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500nucleotides). In some instances, the length of the splintoligonucleotide is from 500 to 1000 nucleotides.

(c) Methods for Increasing Capture Efficiency of a Spatial Array

1. Probe Assembly, Padlock Hybridization, and Padlock Ligation

In some instances, probes are printed on a substrate. Arrays can beprepared by a variety of methods. In some embodiments, arrays areprepared through the synthesis (e.g., in situ synthesis) ofoligonucleotides on the array, or by jet printing or lithography. Forexample, light-directed synthesis of high-density DNA oligonucleotidescan be achieved by photolithography or solid-phase DNA synthesis. Toimplement photolithographic synthesis, synthetic linkers modified withphotochemical protecting groups can be attached to a substrate and thephotochemical protecting groups can be modified using aphotolithographic mask (applied to specific areas of the substrate) andlight, thereby producing an array having localized photo-deprotection.Many of these methods are known in the art, and are described e.g., inMiller et al., “Basic concepts of microarrays and potential applicationsin clinical microbiology.” Clinical Microbiology Reviews 22.4 (2009):611-633; US201314111482A; U.S. Pat. No. 9,593,365B2; US2019203275; andWO2018091676, which are each incorporated herein by reference in itsentirety. Additional disclosure of capture probe assembly is provided inWO 2020/176788 and/or U.S. Patent Application Publication No.2020/0277663, each of which is incorporated by reference in itsentirety.

In some embodiments, the capture probe and/or the splint oligonucleotidecan be used to hybridize the padlock probe. For example, in someembodiments, a method of increasing capture efficiency of a spatialarray includes contacting a substrate including a plurality of captureprobes and a plurality of splint oligonucleotides with a padlock probe;wherein a capture probe of the plurality includes a first dockingsequence, a spatial barcode, and a capture domain; a splintoligonucleotide includes a second docking sequence; the padlock probeincludes a first complementary sequence that is complementary to thefirst docking sequence of the capture probe and a second complementarysequence that is complementary to the second docking sequence of thesecond oligonucleotide; hybridizing the first docking sequence of thecapture probe to the first complementary docking sequence of the padlockprobe and hybridizing the second docking sequence of the splintoligonucleotide to the second complementary docking sequence of thepadlock probe; ligating the 3′ end of the first oligonucleotide to the5′ end of the first oligonucleotide, thereby forming a ligation product;and amplifying the ligation product using rolling circle amplificationto form an amplified ligation product. In some embodiments, theplurality of capture probes are immobilized on the substrate.

In some embodiments, the step of hybridizing the first docking sequenceand the second docking sequence to the first docking padlock sequenceand to the second docking padlock sequence is at a temperature fromabout 50° C. to about 75° C. For example, from about 50° C. to about 70°C., about 50° C. to about 65° C., about 50° C. to about 60° C., about50° C. to about 55° C., about 70° C. to about 75° C., about 65° C. toabout 75° C., about 60° C. to about 75° C., or about 55° C. to about 75°C. In some embodiments, the first temperature is from about 55° C. toabout 70° C., or from about 60° C. to about 65° C.

In some instances, when a splint oligonucleotide is used, the splintoligonucleotide and the padlock probe are added to the array at the sametime. In some instances, the splint oligonucleotide is added prior toaddition of the padlock probe, allowing the splint oligonucleotide firstto hybridize to the capture probe.

After hybridization, in some embodiments, the substrate can be treatedwith one or more enzymes and/or one or more reagents, as describedherein. For example, referring to FIG. 8 (middle panel), one or more DNApolymerase enzymes and dNTPs can be used to fill in the gap between thefirst docking padlock sequence and the second docking padlock sequence(dotted line with arrow).

In some embodiments, a polymerase catalyzes synthesis of a complementarystrand of the ligation product, creating a double-stranded ligationproduct. In some instances, the polymerase is DNA polymerase. In someembodiments, the polymerase has 5′ to 3′ polymerase activity. In someembodiments, the polymerase has 3′ to 5′ exonuclease activity forproofreading. In some embodiments, the polymerase has 5′ to 3′polymerase activity and 3′ to 5′ exonuclease activity for proofreading.

In some instances, the methods further include ligating the two ends ofthe padlock probe. In some instances, the ligation is an enzymaticligation reaction, using a ligase (e.g., T4 RNA ligase (Rnl2), a SplintRligase, a single stranded DNA ligase, or a T4 DNA ligase). See, e.g.,Zhang et al.; RNA Biol. 2017; 14(1): 36-44, which is incorporated byreference in its entirety, for a description of KOD ligase. Followingthe enzymatic ligation reaction, the padlock probe may be consideredligated. In some embodiments, the enzymatic ligation utilizes one ormore of T4 DNA ligase, Tth DNA ligase, Taq DNA ligase, Thermococcus sp.(strain 9oN) DNA ligase, and Ampligase™. Derivatives, e.g.,sequence-modified derivatives, and/or mutants thereof, can also be used.

In some embodiments, a padlock probe may comprise a reactive moiety suchthat, upon hybridization to the target and exposure to appropriateligation conditions, the padlock probe may ligate to itself. In someembodiments, a padlock probe that includes a reactive moiety is ligatedchemically. A reactive moiety of a probe may be selected from thenon-limiting group consisting of azides, alkynes, nitrones (e.g.,1,3-nitrones), strained alkenes (e.g., trans-cycloalkenes such ascyclooctenes or oxanorbornadiene), tetrazines, tetrazoles, iodides,thioates (e.g., phorphorothioate), acids, amines, and phosphates. Forexample, the first reactive moiety of a padlock probe may comprise anazide moiety, and a second reactive moiety of a padlock probe maycomprise an alkyne moiety. The first and second reactive moieties mayreact to form a linking moiety. A reaction between the first and secondreactive moieties may be, for example, a cycloaddition reaction such asa strain-promoted azide-alkyne cycloaddition, a copper-catalyzedazide-alkyne cycloaddition, a strain-promoted alkyne-nitronecycloaddition, a Diels-Alder reaction, a [3+2] cycloaddition, a [4+2]cycloaddition, or a [4+1] cycloaddition; a thiol-ene reaction; anucleophilic substation reaction; or another reaction. In some cases,reaction between the first and second reactive moieties may yield atriazole moiety or an isoxazoline moiety. A reaction between the firstand second reactive moieties may involve subjecting the reactivemoieties to suitable conditions such as a suitable temperature, pH, orpressure and providing one or more reagents or catalysts for thereaction. For example, a reaction between the first and second reactivemoieties may be catalyzed by a copper catalyst, a ruthenium catalyst, ora strained species such as a difluorooctyne, dibenzylcyclooctyne, orbiarylazacyclooctynone. Reaction between a first reactive moiety of afirst probe hybridized to a first target region (e.g., a first targetsequence or first portion) of the nucleic acid molecule and a secondreactive moiety of a third probe oligonucleotide hybridized to a secondtarget region (e.g., a first target sequence or a first portion) of thenucleic acid molecule may link the first probe and the second probe toprovide a ligated probe. Upon linking, the padlock probe may beconsidered ligated. Accordingly, reaction of the first and secondreactive moieties may comprise a chemical ligation reaction such as acopper-catalyzed 5′ azide to 3′ alkyne “click” chemistry reaction toform a triazole linkage between the padlock probe sequences. In othernon-limiting examples, an iodide moiety may be chemically ligated to aphosphorothioate moiety to form a phosphorothioate bond, an acid may beligated to an amine to form an amide bond, and/or a phosphate and aminemay be ligated to form a phosphoramidate bond.

In some embodiments, the methods described herein further includes astep of washing away any oligonucleotides (i.e., splint oligonucleotidesor padlock probes) that do not hybridize to the capture domain. Steps ofwashing to remove unbound oligonucleotides can be performed using any ofthe wash methods described herein or known in the art (e.g., 1×SSC).

2. Padlock Probe Amplification

In some instances, after hybridizing the padlock probe to the captureprobe, the padlock probe (i.e., the ligated padlock probe) is amplified.Amplifying the ligated padlock probe can include an isothermal or anon-isothermal amplification reaction. In some embodiments, amplifyingof the ligation product comprises using rolling circle amplification. Insome embodiments, amplification includes use of a polymerase asdisclosed herein. In some embodiments, amplification includes use of aPhi29 polymerase. In some embodiments, a product of the rolling circleamplification results in the creation of multiple copies of a capturedomain and multiple copies of a barcode.

In some embodiments, the ligated padlock probe comprises a functionalsequence that is capable of binding to a primer used for amplification(referred to herein as the “amplification primer” or “primer used foramplification”). In some embodiments, the amplification primer is usedto amplify the ligated padlock probe, thereby creating multiple copiesof a capture domain.

3. Analyte or Analyte Derivative Hybridization and Spatial Analysis

After generating a plurality of padlock probes from the rolling circleamplification steps, in some instances, the biological sample iscontacted with the substrate. The methods disclosed herein includedetection of analytes that include protein and nucleic acids. In someinstances, the method includes detection of nucleic acids. In someinstances, the methods include detection of proteins.

Referring to FIG. 9, after amplification of the padlock probe, thepadlock probe comprises multiple copies of a capture domain. As shown inFIG. 9, multiple analytes, each of which comprise a sequence (e.g., apoly(A) sequence) complementary to the capture domain, hybridize to theamplified padlock probe. In some instances, the sequence complementaryto the capture domain of the amplified padlock probe is apoly-adenylated (poly(A)) sequence. In some instances, the sequencecomplementary to the capture domain of the amplified padlock probe isdesigned to be specific to a sequence of interest (i.e., in a targetanalyte). In some instances, at least two, at least three, at leastfour, at least five, or more (e.g., 6, 7, 8, 9, 10, 15, 20 or more)analytes hybridize to a single amplified padlock probe.

In some instances, where multiple analytes hybridize to the sameamplified padlock probe, the amplified padlock probe comprises acleavage site so that each interaction between an analyte and thecapture domain of an amplified padlock probe can be analyzedindependently. In some instances, the cleavage site is located such thatthe spatial barcode is cleaved and migrates with the hybridizedanalyte/amplified padlock probe nucleic acid.

In the setting of nucleic acid detection, the nucleic acid analytehybridizes directly to the capture probes on the amplified padlockprobe. In the setting of protein detection, referring to FIG. 4, ananalyte binding moiety 402 comprises a protein-binding moiety 404 and anoligonucleotide 408. In some instances, the analyte binding moieties areadded to the biological sample. After association of the protein withthe protein-binding moiety 404, the oligonucleotide is captured by acapture domain of the padlock probe. Thus, the analyte binding moiety asused herein can be considered an analyte derivate, and itsoligonucleotide can be captured and analyzed in a similar way that anucleic acid analyte can be analyzed. Embodiments for analyte bindingmoieties has been described previously e.g., in WO 2020/176788 and/orU.S. Patent Application Publication No. 2020/0277663, each of which isincorporated by reference in its entirety.

In some embodiments, the methods provided herein include apermeabilizing step in order to release the analytes from the biologicalsample. In some embodiments, permeabilization occurs using a protease.In some embodiments, the protease is an endopeptidase. Endopeptidasesthat can be used include but are not limited to trypsin, chymotrypsin,elastase, thermolysin, pepsin, clostripan, glutamyl endopeptidase(GluC), ArgC, peptidyl-asp endopeptidase (ApsN), endopeptidase LysC andendopeptidase LysN. In some embodiments, the endopeptidase is pepsin. Insome embodiments, after creating the padlock probe copies, thebiological sample is permeabilized. In some embodiments, the biologicalsample is permeabilized contemporaneously with or prior to contactingthe biological sample with a padlock probe.

In some embodiments, methods provided herein include permeabilization ofthe biological sample such that the capture probe can more easily bindto the padlock probe (i.e., compared to no permeabilization). In someembodiments, reverse transcription (RT) reagents can be added topermeabilized biological samples. Incubation with the RT reagents canproduce spatially-barcoded full-length cDNA from the captured analytes(e.g., polyadenylated mRNA). Second strand reagents (e.g., second strandprimers, enzymes) can be added to the biological sample on the substrateto initiate second strand synthesis.

In some instances, the permeabilization step includes application of apermeabilization buffer to the biological sample. In some instances, thepermeabilization buffer includes a buffer (e.g., Tris pH 7.5), MgCl₂,sarkosyl detergent (e.g., sodium lauroyl sarcosinate) or otherdetergent, enzyme (e.g., proteinase K, pepsin, collagenase, etc.), andnuclease free water. In some instances, the permeabilization step isperformed at 37° C. In some instances, the permeabilization step isperformed for about 20 minutes to 2 hours (e.g., about 20 minutes, about30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 1.5hours, or about 2 hours). In some instances, the releasing step isperformed for about 40 minutes.

After permeabilization, in some instances, the analytes are captured bythe capture domains of the padlock probe. In some embodiments, suchmethods of increasing capture efficiency of a spatial array describedherein includes contacting the spatial array with the biological sampleand allowing the analyte to interact with the padlock probes. In someembodiments, the determining step includes amplifying all or part of theanalyte bound to the padlock probes and amplifying all or part of thespatial barcode and the target analyte, or compliments thereof.

In some embodiments, the method includes amplifying all or part of theanalyte using isothermal amplification or non-isothermal amplification.In some embodiments, the amplifying creates an amplifyed product thatincludes (i) all or part of a sequence of the analyte bound to the firstcapture domain and/or the second capture domain, or a complementthereof, and (ii) all or a part of the sequence of the first spatialbarcode and/or the second spatial barcode, or a complement thereof. Insome embodiments, the associating step also includes determining (i) allor part of the sequence of the first spatial barcode and (ii) all orpart of the sequence of the second spatial barcode and using thedetermined sequence of (i) and (ii) to identify the location of theanalyte in the spatial array. In some embodiments, the determining stepincludes sequencing. A non-limiting example of sequencing that can beused to determine the sequence of the analyte and/or spatial barcodes(e.g., first and/or second spatial barcode) is in situ sequencing. Insome embodiments, in situ sequencing is performed viasequencing-by-synthesis (SBS), sequential fluorescence hybridization,sequencing by ligation, nucleic acid hybridization, or high-throughputdigital sequencing techniques. In some embodiments the analyte is RNA orDNA. In some embodiments, the analyte is a protein.

More particularly, after an analyte (e.g., a first analyte, a secondanalyte, etc.) has hybridized or otherwise been associated with thepadlock probe according to any of the methods described above inconnection with the general spatial cell-based analytical methodology,the barcoded constructs that result from hybridization/association areanalyzed.

In some embodiments, after contacting a biological sample with asubstrate that includes capture probes, a removal step can optionally beperformed to remove all or a portion of the biological sample from thesubstrate. In some embodiments, the removal step includes enzymaticand/or chemical degradation of cells of the biological sample. Forexample, the removal step can include treating the biological samplewith an enzyme (e.g., a proteinase, e.g., proteinase K) to remove atleast a portion of the biological sample from the substrate. In someembodiments, the removal step can include ablation of the tissue (e.g.,laser ablation).

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample), the method comprising: (a) optionally stainingand/or imaging a biological sample on a substrate; (b) permeabilizing(e.g., providing a solution comprising a permeabilization reagent to)the biological sample on the substrate; (c) contacting the biologicalsample with an array comprising a plurality of capture probes andpadlock probes, wherein the padlock probe captures the biologicalanalyte; and (d) analyzing the captured biological analyte, therebyspatially detecting the biological analyte; wherein the biologicalsample is fully or partially removed from the substrate.

In some embodiments, a biological sample is not removed from thesubstrate. For example, the biological sample is not removed from thesubstrate prior to releasing a capture probe (e.g., a padlock probe or acapture probe bound to an analyte) from the substrate. In someembodiments, such releasing comprises cleavage of the padlock probe fromthe substrate (e.g., via a cleavage domain). In some embodiments, suchreleasing does not comprise releasing the padlock probe from thesubstrate (e.g., a copy of the padlock probe bound to an analyte can bemade and the copy can be released from the substrate, e.g., viadenaturation). In some embodiments, the biological sample is not removedfrom the substrate prior to analysis of an analyte bound to a padlockprobe after it is released from the substrate. In some embodiments, thebiological sample remains on the substrate during removal of a padlockprobe from the substrate and/or analysis of an analyte bound to thepadlock probe after it is released from the substrate. In someembodiments, the biological sample remains on the substrate duringremoval (e.g., via denaturation) of a copy of the padlock probe (e.g.,complement). In some embodiments, analysis of an analyte bound to apadlock probe from the substrate can be performed without subjecting thebiological sample to enzymatic and/or chemical degradation of the cells(e.g., permeabilized cells) or ablation of the tissue (e.g., laserablation).

In some embodiments, at least a portion of the biological sample is notremoved from the substrate. For example, a portion of the biologicalsample can remain on the substrate prior to releasing a padlock probe(e.g., a padlock probe bound to an analyte) from the substrate and/oranalyzing an analyte bound to a padlock probe released from thesubstrate. In some embodiments, at least a portion of the biologicalsample is not subjected to enzymatic and/or chemical degradation of thecells (e.g., permeabilized cells) or ablation of the tissue (e.g., laserablation) prior to analysis of an analyte bound to a padlock probe fromthe substrate.

In some embodiments, the methods provided herein include spatiallydetecting an analyte (e.g., detecting the location of an analyte, e.g.,a biological analyte) from a biological sample (e.g., present in abiological sample) that include: (a) optionally staining and/or imaginga biological sample on a substrate; (b) permeabilizing (e.g., providinga solution comprising a permeabilization reagent to) the biologicalsample on the substrate; (c) contacting the biological sample with anarray comprising a plurality of capture probes and padlock probes,wherein the padlock probe captures the biological analyte; and (d)analyzing the captured biological analyte, thereby spatially detectingthe biological analyte; where the biological sample is not removed fromthe substrate.

In some embodiments, provided herein are methods for spatially detectinga biological analyte of interest from a biological sample that include:(a) staining and imaging a biological sample on a substrate; (b)providing a solution comprising a permeabilization reagent to thebiological sample on the substrate; (c) contacting the biological samplewith an array on a substrate, wherein the array comprises one or morecapture probe pluralities thereby allowing the one or more pluralitiesof capture probes to capture the biological analyte of interest; and (d)analyzing the captured biological analyte, thereby spatially detectingthe biological analyte of interest; where the biological sample is notremoved from the substrate.

In some embodiments, the method further includes subjecting a region ofinterest in the biological sample to spatial transcriptomic analysis. Insome embodiments, one or more of the padlock probes includes a capturedomain. In some embodiments, one or more of the padlock probes comprisesa unique molecular identifier (UMI). In some embodiments, one or more ofthe padlock probes comprises a cleavage domain. In some embodiments, thecleavage domain comprises a sequence recognized and cleaved byuracil-DNA glycosylase, apurinic/apyrimidinic (AP) endonuclease (APE1),uracil-specific excision reagent (USER), and/or an endonuclease VIII. Insome embodiments, one or more padlock probes do not comprise a cleavagedomain and is not cleaved from the array.

In some instances, the padlock probe can be cleaved, separating eachinteraction between an analyte and the padlock probe.

In some embodiments, the cleaved padlock probe can be extended (an“extended padlock probe,” e.g., as described herein). For example,extending a padlock probe can include generating cDNA from a captured(hybridized) RNA. This process involves synthesis of a complementarystrand of the hybridized nucleic acid, e.g., generating cDNA based onthe captured RNA template (the RNA hybridized to the capture domain ofthe padlock probe). Thus, in an initial step of extending a padlockprobe, e.g., the cDNA generation, the captured (hybridized) nucleicacid, e.g., RNA, acts as a template for the extension, e.g., a reversetranscription step.

In some embodiments, the padlock probe is extended using reversetranscription. For example, reverse transcription includes synthesizingcDNA (complementary or copy DNA) from RNA, e.g., (messenger RNA), usinga reverse transcriptase. In some embodiments, reverse transcription isperformed while the tissue is still in place, generating an analytelibrary, where the analyte library includes the spatial barcodes fromthe adjacent padlock probes. In some embodiments, the padlock probe isextended using one or more DNA polymerases.

In some embodiments, a padlock probe includes a primer for producing thecomplementary strand of a nucleic acid hybridized to the padlock probe,e.g., a primer for DNA polymerase and/or reverse transcription. Thenucleic acid, e.g., DNA and/or cDNA, molecules generated by theextension reaction incorporate the sequence of the padlock probe. Theextension of the padlock probe, e.g., a DNA polymerase and/or reversetranscription reaction, can be performed using a variety of suitableenzymes and protocols.

In some embodiments, a full-length DNA (e.g., cDNA) molecule isgenerated. In some embodiments, a “full-length” DNA molecule refers tothe whole of the captured nucleic acid molecule. However, if a nucleicacid (e.g., RNA) was partially degraded in the tissue sample, then thecaptured nucleic acid molecules will not be the same length as theinitial RNA in the tissue sample. In some embodiments, the 3′ end of theextended probes, e.g., first strand cDNA molecules, is modified. Forexample, a linker or adaptor can be ligated to the 3′ end of theextended probes. This can be achieved using single stranded ligationenzymes such as T4 RNA ligase or Circligase™ (available from Lucigen,Middleton, Wis.). In some embodiments, template switchingoligonucleotides are used to extend cDNA in order to generate afull-length cDNA (or as close to a full-length cDNA as possible). Insome embodiments, a second strand synthesis helper probe (a partiallydouble stranded DNA molecule capable of hybridizing to the 3′ end of theextended padlock probe), can be ligated to the 3′ end of the extendedprobe, e.g., first strand cDNA, molecule using a double strandedligation enzyme such as T4 DNA ligase. Other enzymes appropriate for theligation step are known in the art and include, e.g., Tth DNA ligase,Taq DNA ligase, Thermococcus sp. (strain 9° N) DNA ligase (9° N™ DNAligase, New England Biolabs), Ampligase™ (available from Lucigen,Middleton, Wis.), and SplintR (available from New England Biolabs,Ipswich, Mass.). In some embodiments, a polynucleotide tail, e.g., apoly(A) tail, is incorporated at the 3′ end of the extended probemolecules. In some embodiments, the polynucleotide tail is incorporatedusing a terminal transferase active enzyme.

In some embodiments, double-stranded extended padlock probes are treatedto remove any unextended padlock probes prior to amplification and/oranalysis, e.g., sequence analysis. This can be achieved by a variety ofmethods, e.g., using an enzyme to degrade the unextended probes, such asan exonuclease enzyme, or purification columns.

In some embodiments, extended padlock probes are amplified to yieldquantities that are sufficient for analysis, e.g., via DNA sequencing.In some embodiments, the first strand of the extended padlock probes(e.g., DNA and/or cDNA molecules) acts as a template for theamplification reaction (e.g., a polymerase chain reaction).

In some embodiments, the amplification reaction incorporates an affinitygroup onto the extended padlock probe (e.g., RNA-cDNA hybrid) using aprimer including the affinity group. In some embodiments, the primerincludes an affinity group and the extended padlock probes includes theaffinity group. The affinity group can correspond to any of the affinitygroups described previously.

In some embodiments, the extended padlock probes including the affinitygroup can be coupled to a substrate specific for the affinity group. Insome embodiments, the substrate can include an antibody or antibodyfragment. In some embodiments, the substrate includes avidin orstreptavidin and the affinity group includes biotin. In someembodiments, the substrate includes maltose and the affinity groupincludes maltose-binding protein. In some embodiments, the substrateincludes maltose-binding protein and the affinity group includesmaltose. In some embodiments, amplifying the extended padlock probes canfunction to release the extended probes from the surface of thesubstrate, insofar as copies of the extended probes are not immobilizedon the substrate.

In some embodiments, the extended padlock probe or complement oramplicon thereof is released. The step of releasing the extended padlockprobe or complement or amplicon thereof from the surface of thesubstrate can be achieved in a number of ways. In some embodiments, anextended padlock probe or a complement thereof is released from thearray by nucleic acid cleavage and/or by denaturation (e.g., by heatingto denature a double-stranded molecule).

In some embodiments, the extended padlock probe or complement oramplicon thereof is released from the surface of the substrate (e.g.,array) by physical means. For example, where the extended padlock probeis indirectly immobilized on the array substrate, e.g., viahybridization to a surface probe, it can be sufficient to disrupt theinteraction between the extended padlock probe and the surface probe.Methods for disrupting the interaction between nucleic acid moleculesinclude denaturing double stranded nucleic acid molecules are known inthe art. A straightforward method for releasing the DNA molecules (i.e.,of stripping the array of extended probes) is to use a solution thatinterferes with the hydrogen bonds of the double stranded molecules. Insome embodiments, the extended padlock probe is released by an applyingheated solution, such as water or buffer, of at least 85° C., e.g., atleast 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99° C. In some embodiments,a solution including salts, surfactants, etc. that can furtherdestabilize the interaction between the nucleic acid molecules is addedto release the extended padlock probe from the substrate.

In some embodiments, where the extended padlock probe includes acleavage domain, the extended padlock probe is released from the surfaceof the substrate by cleavage. For example, the cleavage domain of theextended padlock probe can be cleaved by any of the methods describedherein. In some embodiments, the extended padlock probe is released fromthe surface of the substrate, e.g., via cleavage of a cleavage domain inthe extended padlock probe, prior to the step of amplifying the extendedpadlock probe.

In some embodiments, probes complementary to the extended padlock probecan be contacted with the substrate. In some embodiments, the biologicalsample can be in contact with the substrate when the probes arecontacted with the substrate. In some embodiments, the biological samplecan be removed from the substrate prior to contacting the substrate withprobes. In some embodiments, the probes can be labeled with a detectablelabel (e.g., any of the detectable labels described herein). In someembodiments, probes that do not specially bind (e.g., hybridize) to anextended padlock probe can be washed away. In some embodiments, probescomplementary to the extended padlock probe can be detected on thesubstrate (e.g., imaging, any of the detection methods describedherein).

In some embodiments, probes complementary to an extended padlock probecan be about 4 nucleotides to about 100 nucleotides long. In someembodiments, probes (e.g., detectable probes) complementary to anextended padlock probe can be about 10 nucleotides to about 90nucleotides long. In some embodiments, probes (e.g., detectable probes)complementary to an extended padlock probe can be about 20 nucleotidesto about 80 nucleotides long. In some embodiments, probes (e.g.,detectable probes) complementary to an extended padlock probe can beabout 30 nucleotides to about 60 nucleotides long. In some embodiments,probes (e.g., detectable probes) complementary to an extended padlockprobe can be about 40 nucleotides to about 50 nucleotides long. In someembodiments, probes (e.g., detectable probes) complementary to anextended padlock probe can be about 5, about 6, about 7, about 8, about9, about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24, about 25, about 26, about 27, about 28, about 29, about 30,about 31, about 32, about 33, about 34, about 35, about 36, about 37,about 38, about 39, about 40, about 41, about 42, about 43, about 44,about 45, about 46, about 47, about 48, about 49, about 50, about 51,about 52, about 53, about 54, about 55, about 56, about 57, about 58,about 59, about 60, about 61, about 62, about 63, about 64, about 65,about 66, about 67, about 68, about 69, about 70, about 71, about 72,about 73, about 74, about 75, about 76, about 77, about 78, about 79,about 80, about 81, about 82, about 83, about 84, about 85, about 86,about 87, about 88, about 89, about 90, about 91, about 92, about 93,about 94, about 95, about 96, about 97, about 98, and about 99nucleotides long.

In some embodiments, about 1 to about 100 probes can be contacted to thesubstrate and specifically bind (e.g., hybridize) to an extended padlockprobe. In some embodiments, about 1 to about 10 probes can be contactedto the substrate and specifically bind (e.g., hybridize) to an extendedpadlock probe. In some embodiments, about 10 to about 100 probes can becontacted to the substrate and specifically bind (e.g., hybridize) to anextended padlock probe. In some embodiments, about 20 to about 90 probescan be contacted to the substrate and specifically bind (e.g.,hybridize) to an extended padlock probe. In some embodiments, about 30to about 80 probes (e.g., detectable probes) can be contacted to thesubstrate and specifically bind (e.g., hybridize) to an extended padlockprobe. In some embodiments, about 40 to about 70 probes can be contactedto the substrate and specifically bind (e.g., hybridize) to an extendedpadlock probe. In some embodiments, about 50 to about 60 probes can becontacted to the substrate and specifically bind (e.g., hybridize) to anextended padlock probe. In some embodiments, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 11, about12, about 13, about 14, about 15, about 16, about 17, about 18, about19, about 20, about 21, about 22, about 23, about 24, about 25, about26, about 27, about 28, about 29, about 30, about 31, about 32, about33, about 34, about 35, about 36, about 37, about 38, about 39, about40, about 41, about 42, about 43, about 44, about 45, about 46, about47, about 48, about 49, about 50, about 51, about 52, about 53, about54, about 55, about 56, about 57, about 58, about 59, about 60, about61, about 62, about 63, about 64, about 65, about 66, about 67, about68, about 69, about 70, about 71, about 72, about 73, about 74, about75, about 76, about 77, about 78, about 79, about 80, about 81, about82, about 83, about 84, about 85, about 86, about 87, about 88, about89, about 90, about 91, about 92, about 93, about 94, about 95, about96, about 97, about 98, and about 99 probes can be contacted to thesubstrate and specifically bind (e.g., hybridize) to an extended capturepadlock.

In some embodiments, the probes can be complementary to a single analyte(e.g., a single gene). In some embodiments, the probes can becomplementary to one or more analytes (e.g., analytes in a family ofgenes). In some embodiments, the probes (e.g., detectable probes) can befor a panel of genes associated with a disease (e.g., cancer,Alzheimer's disease, Parkinson's disease).

In some instances, the padlock probe can be amplified or copied,creating a plurality of cDNA molecules. In some embodiments, cDNA can bedenatured from the padlock probe template and transferred (e.g., to aclean tube) for amplification, and/or library construction. Thespatially-barcoded cDNA can be amplified via PCR prior to libraryconstruction. The cDNA can then be enzymatically fragmented andsize-selected in order to optimize for cDNA amplicon size. P5 and P7sequences directed to capturing the amplicons on a sequencing flowcell(e.g., Illumina sequencing instruments) can be appended to theamplicons, i7, and i5 can be used as sample indexes, and TruSeq Read 2can be added via End Repair, A-tailing, Adaptor Ligation, and PCR. ThecDNA fragments can then be sequenced using paired-end sequencing usingTruSeq Read 1 and TruSeq Read 2 as sequencing primer sites. A skilledartisan will understand that additional or alternative sequences used byother sequencing instruments or technologies are also equally applicablefor use in the aforementioned methods as the current methods are notlimited to any a particular sequencing platform.

In some embodiments, where a sample is barcoded directly viahybridization with padlock probes or analyte capture agents hybridized,bound, or associated with either the cell surface, or introduced intothe cell, as described above, sequencing can be performed on the intactsample.

A wide variety of different sequencing methods can be used to analyzethe barcoded analyte or moiety. In general, sequenced polynucleotidescan be, for example, nucleic acid molecules such as deoxyribonucleicacid (DNA) or ribonucleic acid (RNA), including variants or derivativesthereof (e.g., single stranded DNA or DNA/RNA hybrids, and nucleic acidmolecules with a nucleotide analog).

Sequencing of polynucleotides can be performed by various systems. Moregenerally, sequencing can be performed using nucleic acid amplification,polymerase chain reaction (PCR) (e.g., digital PCR and droplet digitalPCR (ddPCR), quantitative PCR, real time PCR, multiplex PCR, PCR-basedsingle plex methods, emulsion PCR), and/or isothermal amplification.Non-limiting examples of methods for sequencing genetic materialinclude, but are not limited to, DNA hybridization methods (e.g.,Southern blotting), restriction enzyme digestion methods, Sangersequencing methods, next-generation sequencing methods (e.g.,single-molecule real-time sequencing, sequence by synthesis sequencing,nanopore sequencing, and Polony sequencing), ligation methods, andmicroarray methods.

In some embodiments, a capture domain as described herein (e.g., on apadlock probe) is blocked, thereby preventing an unwanted hybridizationto the capture domain. In some embodiments, a blocking probe or chemicalmoiety is used to block or modify the free 3′ end of the padlock probecapture domain. In some embodiments, a blocking probe can be hybridizedto the padlock probe capture domain of the second probe to mask the free3′ end of the padlock probe capture domain. In some embodiments, ablocking probe can be a hairpin probe or partially double strandedprobe. In some embodiments, the free 3′ end of the padlock probe capturedomain of the second probe can be blocked by chemical modification,e.g., addition of an azidomethyl group as a chemically reversiblecapping moiety such that the capture probes do not include a free 3′end. Blocking or modifying the padlock probe capture domain,particularly at the free 3′ end of the padlock probe capture domain,prior to contacting second probe with the substrate, preventshybridization of the second probe to the capture domain (e.g., preventsthe capture of a poly(A) of a padlock probe capture domain to a poly(T)capture domain). In some embodiments, a blocking probe can be referredto as a padlock probe capture domain blocking moiety.

(d) Biological Sample, Analytes and Sample Preparation

1. Biological Samples and Analytes

Methods disclosed herein can be performed on any type of sample. In someembodiments, the sample is a fresh tissue sample. In some instances, thebiological sample is a tissue, a tissue section, an organ, an organism,or a cell culture sample. In some instances, the biological sample is aformalin-fixed, paraffin-embedded (FFPE) sample, a frozen sample, or afresh sample.

In some embodiments, the analyte includes one or more of RNA, DNA, aprotein, a small molecule, and a metabolite. In some embodiments, theanalyte (e.g., target analyte) is a single-stranded oligonucleotide. Insome embodiments, the single-stranded oligonucleotide is RNA. In someembodiments, the RNA is mRNA. In some embodiments, the mRNA is an mRNAof interest. In some embodiments, the multiple target analytes aredetected. The multiple targets can, in some instances, include sequencesthat have at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to eachother. In some instances, the multiple targets each include one or moreconserved sequences. In some instances, the multiple targets are mRNAsthat encode for proteins that have a similar function. In someinstances, the multiple targets are mRNAs that encode for proteins thatfunction in the same or a similar cellular pathway.

Subjects from which biological samples can be obtained can be healthy orasymptomatic individuals that have or are suspected of having a disease(e.g., cancer) or a pre-disposition to a disease, and/or individualsthat are in need of therapy or suspected of needing therapy. In someinstances, the biological sample can include one or more diseased cells.A diseased cell can have altered metabolic properties, gene expression,protein expression, and/or morphologic features. Examples of diseasesinclude inflammatory disorders, metabolic disorders, nervous systemdisorders, and cancer. In some instances, the biological sample includescancer or tumor cells. Cancer cells can be derived from solid tumors,hematological malignancies, cell lines, or obtained as circulating tumorcells. In some instances, the biological sample is a heterogenoussample. In some instances, the biological sample is a heterogenoussample that includes tumor or cancer cells and/or stromal cells,

In some embodiments, the biological sample is from a human subject.

FFPE samples generally are heavily cross-linked and fragmented, andtherefore this type of sample allows for limited RNA recovery usingconventional detection techniques. In certain embodiments, methods oftargeted RNA capture provided herein are less affected by RNAdegradation associated with FFPE fixation than other methods (e.g.,methods that take advantage of poly(T) capture and reverse transcriptionof mRNA). In certain embodiments, methods provided herein enablesensitive measurement of specific genes of interest that otherwise mightbe missed with a whole transcriptomic approach.

In some instances, FFPE samples are stained (e.g., using H&E,immunofluorescence, etc.). The methods disclosed herein are compatiblewith staining methods that will allow for morphological context overlaidwith transcriptomic analysis. However, depending on the need somesamples may be stained with only a nuclear stain, such as staining asample with only hematoxylin and not eosin, the use of DAPI, etc. whenlocation of a cell nucleus is needed.

In some embodiments, a biological sample (e.g. tissue section) can befixed with methanol, stained with hematoxylin and eosin, and imaged. Insome embodiments, fixing, staining, and imaging occurs before one ormore probes are hybridized to the sample. Some embodiments of any of theworkflows described herein can further include a destaining step (e.g.,a hematoxylin and eosin destaining step), after imaging of the sampleand prior to permeabilizing the sample. For example, destaining can beperformed by performing one or more (e.g., one, two, three, four, orfive) washing steps (e.g., one or more (e.g., one, two, three, four, orfive) washing steps performed using a buffer including HCl). The imagescan be used to map spatial gene expression patterns back to thebiological sample. A permeabilization enzyme can be used to permeabilizethe biological sample directly on the substrate.

In some embodiments, the FFPE sample is deparaffinized, permeabilized,equilibrated, and blocked before target probe oligonucleotides areadded. In some embodiments, deparaffinization includes the use ofxylenes. In some embodiments, deparaffinization includes multiple washeswith xylenes. In some embodiments, deparaffinization includes multiplewashes with xylenes followed by removal of xylenes using multiple roundsof graded alcohol washes followed by washing the sample with water. Insome aspects, the water is deionized water. In some embodiments,equilibrating and blocking includes incubating the sample in a pre-Hybbuffer. In some embodiments, the pre-Hyb buffer includes yeast tRNA. Insome embodiments, permeabilizing a sample includes washing the samplewith a phosphate buffer. In some embodiments, the buffer is PBS. In someembodiments, the buffer is PBST.

2. Imaging and Staining

In some instances, biological samples can be stained using a widevariety of stains and staining techniques. In some instances, thebiological sample is a section of a tissue (e.g., a 10 μm section). Insome instances, the biological sample is dried after placement onto aglass slide. In some instances, the biological sample is dried at 42° C.In some instances, drying occurs for about 1 hour, about 2, hours, about3 hours, or until the sections become transparent. In some instances,the biological sample can be dried overnight (e.g., in a desiccator atroom temperature).

In some embodiments, a sample can be stained using any number ofbiological stains, including but not limited to, acridine orange,Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin,ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine,methyl green, methylene blue, neutral red, Nile blue, Nile red, osmiumtetroxide, propidium iodide, rhodamine, or safranin. In some instances,the methods disclosed herein include imaging the biological sample. Insome instances, imaging the sample occurs prior to deaminating thebiological sample. In some instances, the sample can be stained usingknown staining techniques, including Can-Grunwald, Giemsa, hematoxylinand eosin (H&E), Jenner's, Leishman, Masson's trichrome, Papanicolaou,Romanowsky, silver, Sudan, Wright's, and/or Periodic Acid Schiff (PAS)staining techniques. PAS staining is typically performed after formalinor acetone fixation. In some instances, the stain is an H&E stain.

In some embodiments, the biological sample can be stained using adetectable label (e.g., radioisotopes, fluorophores, chemiluminescentcompounds, bioluminescent compounds, and dyes) as described elsewhereherein. In some embodiments, a biological sample is stained using onlyone type of stain or one technique. In some embodiments, stainingincludes biological staining techniques such as H&E staining. In someembodiments, staining includes identifying analytes usingfluorescently-conjugated antibodies. In some embodiments, a biologicalsample is stained using two or more different types of stains, or two ormore different staining techniques. For example, a biological sample canbe prepared by staining and imaging using one technique (e.g., H&Estaining and brightfield imaging), followed by staining and imagingusing another technique (e.g., IHC/IF staining and fluorescencemicroscopy) for the same biological sample.

In some embodiments, biological samples can be destained. Methods ofdestaining or decoloring a biological sample are known in the art, andgenerally depend on the nature of the stain(s) applied to the sample.For example, H&E staining can be destained by washing the sample in HCl,or any other acid (e.g., selenic acid, sulfuric acid, hydroiodic acid,benzoic acid, carbonic acid, malic acid, phosphoric acid, oxalic acid,succinic acid, salicylic acid, tartaric acid, sulfurous acid,trichloroacetic acid, hydrobromic acid, hydrochloric acid, nitric acid,orthophosphoric acid, arsenic acid, selenous acid, chromic acid, citricacid, hydrofluoric acid, nitrous acid, isocyanic acid, formic acid,hydrogen selenide, molybdic acid, lactic acid, acetic acid, carbonicacid, hydrogen sulfide, or combinations thereof). In some embodiments,destaining can include 1, 2, 3, 4, 5, or more washes in an acid (e.g.,HCl). In some embodiments, destaining can include adding HCl to adownstream solution (e.g., permeabilization solution). In someembodiments, destaining can include dissolving an enzyme used in thedisclosed methods (e.g., pepsin) in an acid (e.g., HCl) solution. Insome embodiments, after destaining hematoxylin with an acid, otherreagents can be added to the destaining solution to raise the pH for usein other applications. For example, SDS can be added to an aciddestaining solution in order to raise the pH as compared to the aciddestaining solution alone. As another example, in some embodiments, oneor more immunofluorescence stains are applied to the sample via antibodycoupling. Such stains can be removed using techniques such as cleavageof disulfide linkages via treatment with a reducing agent and detergentwashing, chaotropic salt treatment, treatment with antigen retrievalsolution, and treatment with an acidic glycine buffer. Methods formultiplexed staining and destaining are described, for example, inBolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin etal., Nat Commun. 2015; 6:8390, Pirici et al., J. Histochem. Cytochem.2009; 57:567-75, and Glass et al., J. Histochem. Cytochem. 2009;57:899-905, the entire contents of each of which are incorporated hereinby reference.

In some embodiments, immunofluorescence or immunohistochemistryprotocols (direct and indirect staining techniques) can be performed asa part of, or in addition to, the exemplary spatial workflows presentedherein. For example, tissue sections can be fixed according to methodsdescribed herein. The biological sample can be transferred to an array(e.g., capture probe array), wherein analytes (e.g., proteins) areprobed using immunofluorescence protocols. For example, the sample canbe rehydrated, blocked, and permeabilized (3×SSC, 2% BSA, 0.1% Triton X,1 U/μ1 RNAse inhibitor for 10 minutes at 4° C.) before being stainedwith fluorescent primary antibodies (1:100 in 3×SSC, 2% BSA, 0.1% TritonX, 1 U/μ1 RNAse inhibitor for 30 minutes at 4° C.). The biologicalsample can be washed, coverslipped (in glycerol+1 U/μ1 RNAse inhibitor),imaged (e.g., using a confocal microscope or other apparatus capable offluorescent detection), washed, and processed according to analytecapture or spatial workflows described herein.

In some instances, a glycerol solution and a cover slip can be added tothe sample. In some instances, the glycerol solution can include acounterstain (e.g., DAPI).

As used herein, an antigen retrieval buffer can improve antibody capturein IF/IHC protocols. An exemplary protocol for antigen retrieval can bepreheating the antigen retrieval buffer (e.g., to 95° C.), immersing thebiological sample in the heated antigen retrieval buffer for apredetermined time, and then removing the biological sample from theantigen retrieval buffer and washing the biological sample.

In some embodiments, optimizing permeabilization can be useful foridentifying intracellular analytes. Permeabilization optimization caninclude selection of permeabilization agents, concentration ofpermeabilization agents, and permeabilization duration. Tissuepermeabilization is discussed elsewhere herein.

In some embodiments, blocking an array and/or a biological sample inpreparation of labeling the biological sample decreases nonspecificbinding of the antibodies to the array and/or biological sample(decreases background). Some embodiments provide for blockingbuffers/blocking solutions that can be applied before and/or duringapplication of the label, wherein the blocking buffer can include ablocking agent, and optionally a surfactant and/or a salt solution. Insome embodiments, a blocking agent can be bovine serum albumin (BSA),serum, gelatin (e.g., fish gelatin), milk (e.g., non-fat dry milk),casein, polyethylene glycol (PEG), polyvinyl alcohol (PVA), orpolyvinylpyrrolidone (PVP), biotin blocking reagent, a peroxidaseblocking reagent, levamisole, Carnoy's solution, glycine, lysine, sodiumborohydride, pontamine sky blue, Sudan Black, trypan blue, FITC blockingagent, and/or acetic acid. The blocking buffer/blocking solution can beapplied to the array and/or biological sample prior to and/or duringlabeling (e.g., application of fluorophore-conjugated antibodies) to thebiological sample.

3. Preparation of Sample for Application of Probes

In some instances, the biological sample is deparaffinized.Deparaffinization can be achieved using any method known in the art. Forexample, in some instances, the biological samples is treated with aseries of washes that include xylene and various concentrations ofethanol. In some instances, methods of deparaffinization includetreatment with xylene (e.g., three washes at 5 minutes each). In someinstances, the methods further include treatment with ethanol (e.g.,100% ethanol, two washes 10 minutes each; 95% ethanol, two washes 10minutes each; 70% ethanol, two washes 10 minutes each; 50% ethanol, twowashes 10 minutes each). In some instances, after ethanol washes, thebiological sample can be washed with deionized water (e.g., two washesfor 5 minutes each). It is appreciated that one skilled in the art canadjust these methods to optimize deparaffinization.

In some instances, the biological sample is decrosslinked. In someinstances, the biological sample is decrosslinked in a solutioncontaining TE buffer (comprising Tris and EDTA). In some instances, theTE buffer is basic (e.g., at a pH of about 9). In some instances,decrosslinking occurs at about 50° C. to about 80° C. In some instances,decrosslinking occurs at about 70° C. In some instances, decrosslinkingoccurs for about 1 hour at 70° C. Just prior to decrosslinking, thebiological sample can be treated with an acid (e.g., 0.1M HCl for about1 minute). After the decrosslinking step, the biological sample can bewashed (e.g., with 1×PBST).

In some instances, the methods of preparing a biological sample forprobe application include permeabilizing the sample. In some instances,the biological sample is permeabilized using a phosphate buffer. In someinstances, the phosphate buffer is PBS (e.g., lx PBS). In someinstances, the phosphate buffer is PBST (e.g., lx PBST). In someinstances, the permeabilization step is performed multiple times (e.g.,3 times at 5 minutes each).

In some instances, the methods of preparing a biological sample forprobe application include steps of equilibrating and blocking thebiological sample. In some instances, equilibrating is performed using apre-hybridization (pre-Hyb) buffer. In some instances, the pre-Hybbuffer is RNase-free. In some instances, the pre-Hyb buffer contains nobovine serum albumin (BSA), solutions like Denhardt's, or otherpotentially nuclease-contaminated biological materials.

In some instances, the equilibrating step is performed multiple times(e.g., 2 times at 5 minutes each; 3 times at 5 minutes each). In someinstances, the biological sample is blocked with a blocking buffer. Insome instances, the blocking buffer includes a carrier such as tRNA, forexample yeast tRNA such as from brewer's yeast (e.g., at a finalconcentration of 10-20 μg/mL). In some instances, blocking can beperformed for 5, 10, 15, 20, 25, or 30 minutes.

Any of the foregoing steps can be optimized for performance. Forexample, one can vary the temperature. In some instances, thepre-hybridization methods are performed at room temperature. In someinstances, the pre-hybridization methods are performed at 4° C. (in someinstances, varying the timeframes provided herein).

(e) Kits

In some embodiments, also provided herein are kits that include one ormore reagents to detect one or more analytes described herein. In someinstances, the kit includes a substrate comprising a plurality ofcapture probes comprising a capture domain. In some instances, the kitincludes a plurality of probes (e.g., capture probes, padlock probes,and splint oligonucleotides) as described herein.

A non-limiting example of a kit used to perform any of the methodsdescribed herein includes: (a) a substrate comprising a plurality ofcapture probes comprising a spatial barcode and a capture domain; (b) asystem comprising padlock probes and splint oligonucleotides; and (c)instructions for performing the methods provided herein.

Another non-limiting example of a kit used to perform any of the methodsdescribed herein includes: (a) an array comprising a plurality ofcapture probes; (b) padlock probes and splint oligonucleotides; (c) aplurality of enzymes, including a polymerase and a ligase; and (d)instructions for performing the methods provided herein.

EXAMPLES Example 1. Method for Increasing Capture Efficiency of aSpatial Array Using a Padlock Probe

A substrate with a plurality of capture probes is contacted with a firstoligonucleotide. A capture probe of the plurality has a first dockingsequence, a second docking sequence, a barcode, and a capture domain.The first oligonucleotide has a first complementary sequencecomplementary to the first docking sequence of the capture probe and asecond complementary sequence complementary to the second dockingsequence of the capture probe. The first docking sequence of the captureprobe is hybridized to the first complementary sequence of the firstoligonucleotide and the second docking sequence of the capture probe ishybridized to the second complementary sequence of the firstoligonucleotide. The 3′ end of the first oligonucleotide is ligated tothe 5′ end of the first oligonucleotide, thereby forming a ligationproduct; and the ligation product is amplified using rolling circleamplification.

Example 2. Method for Increasing Capture Efficiency of a Spatial ArrayUsing a Padlock Probe and a Splint Oligonucleotide

A substrate with a plurality of capture probes is contacted with a firstoligonucleotide and a second oligonucleotide. A capture probe of theplurality has a first docking sequence, a barcode, and a capture domain.The second oligonucleotide has a second docking sequence, and the firstoligonucleotide includes a first complementary sequence that iscomplementary to the first docking sequence of the capture probe and asecond complementary sequence that is complementary to the seconddocking sequence of the second oligonucleotide. The first dockingsequence of the capture probe is hybridized to the first complementarydocking sequence of the first oligonucleotide, and the second dockingsequence of the second oligonucleotide is hybridized to the secondcomplementary docking sequence of the first oligonucleotide. The 3′ endof the first oligonucleotide is ligated to the 5′ end of the firstoligonucleotide, thereby forming a ligation product. The ligationproduct is amplified using rolling circle amplification to form anamplified ligation product.

Example 3. Method for Increasing Capture Efficiency of a Spatial ArrayUsing an Extended Padlock Probe

A substrate having a plurality of capture probes is contacted with afirst oligonucleotide. A capture probe of the plurality has a firstdocking sequence, a second docking sequence, a barcode, and a capturedomain. The first oligonucleotide has a first complementary sequencecomplementary to the first docking sequence of the capture probe, and asecond complementary sequence complementary to the first dockingsequence of the capture probe. The first docking sequence and the seconddocking sequence of the capture probe are hybridized to the firstcomplementary docking sequence and the second complementary dockingsequence of the oligonucleotide. The first oligonucleotide is extended.The extended 3′ end of the first oligonucleotide is ligated to the 5′end of the first oligonucleotide to form a ligation product; and theligation product is amplified using rolling circle amplification to forman amplified ligation product.

What is claimed is:
 1. A method for preparing a spatial array, themethod comprising: (a) contacting a substrate comprising a plurality ofcapture probes with a padlock probe, wherein (i) a capture probe of theplurality of capture probes comprises a first docking sequence, aspatial barcode, a capture domain, and a second docking sequence; and(ii) the padlock probe comprises: a first docking padlock sequence atits 3′ end that is complementary to the first docking sequence of thecapture probe, and a second docking padlock sequence at its 5′ end thatis complementary to the second docking sequence of the capture probe;(b) hybridizing the first docking sequence to the first docking padlocksequence and hybridizing the second docking sequence to the seconddocking padlock sequence; (c) ligating the padlock probe, therebygenerating a ligated padlock probe that comprises a capture domain; and(d) amplifying the ligated padlock probe, thereby generating a padlockprobe sequence with multiple copies of the capture domain.
 2. The methodof claim 1, wherein the spatial barcode and the capture domain arelocated between the first docking sequence and the second dockingsequence of the capture probe.
 3. The method of claim 1, wherein theligated padlock probe further comprises a primer sequence, or acomplement thereof, and a spatial domain, or a complement thereof. 4.The method of claim 1, wherein the amplifying comprises rolling circleamplification.
 5. The method of claim 4, wherein the padlock probecomprises a 3′OH group, wherein the 3′OH group is a primer for therolling circle amplification.
 6. The method of claim 1, wherein step (b)utilizes a splint oligonucleotide that comprises a sequencecomplementary to the capture probe and a sequence complementary to thepadlock probe.
 7. The method of claim 1, further comprising extendingthe padlock probe via a nucleic acid extension reaction using thecapture probe as a template prior to step (c).
 8. The method of claim 1,wherein the capture domain comprises a poly(T) sequence, a randomsequence, a semi-random sequence or a fixed sequence.
 9. The method ofclaim 1, wherein the ligation step comprises using enzymatic ligation orchemical ligation.
 10. The method of claim 9, wherein the enzymaticligation utilizes a ligase, wherein the ligase is one or more of a T4RNA ligase (Rnl2), a splintR ligase, a single stranded DNA ligase, or aT4 DNA ligase.
 11. The method of claim 1, further comprising spatiallyprofiling an analyte in a biological sample by the steps of: (e)contacting the spatial array with the biological sample; hybridizing theanalyte or analyte derivative to a capture domain of the multiple copiesof the capture domain; and (g) determining (i) all or a part of thesequence of the analyte or analyte derivative, or a complement thereof,and (ii) all or a part of the sequence of the spatial barcode, or acomplement thereof, and using the determined sequence of (i) and (ii) todetermine the abundance and the location of the analyte in thebiological sample.
 12. The method of claim 11, wherein the methodfurther comprises contacting the biological sample with apermeabilization agent, wherein the permeabilization agent is selectedfrom an organic solvent, a detergent, and an enzyme, or a combinationthereof.
 13. The method of claim 11, wherein the determining stepcomprises sequencing.
 14. The method of claim 11, further comprisingimaging the biological sample.
 15. The method of claim 11, wherein thebiological sample is a formalin-fixed, paraffin-embedded (FFPE) sample,a frozen sample, or a fresh sample.
 16. The method of claim 11, whereinthe analyte is an RNA molecule or a protein, or both.
 17. A kitcomprising: (a) an array comprising a plurality of primers; (b) aplurality of padlock probes; (c) a plurality of enzymes comprising apolymerase and a ligase; and (d) instructions for performing the methodof claim
 12. 18. A composition comprising: (a) a substrate comprising aplurality of capture probes, wherein a capture probe of the plurality ofcapture probes comprises a first docking sequence, a spatial barcode, acapture domain, and a second docking sequence; (b) a plurality ofamplified padlock probes, wherein an amplified padlock probe of theplurality of amplified padlock probes comprises: (i) a first dockingpadlock sequence that is complementary to the first docking sequence ofthe capture probe, (ii) a second docking padlock sequence that iscomplementary to the second docking sequence of the capture probe, and(iii) a sequence complementary to the capture domain; and wherein theamplified padlock probe is hybridized to the capture probe.
 19. Thecomposition of claim 18, further comprising an analyte hybridized to theamplified padlock probe.
 20. The composition of claim 18, wherein theamplified padlock probe further comprises a primer sequence, or acomplement thereof, and a spatial domain, or a complement thereof.